Liposomal mitigation of drug-induced inhibition of the cardiac IKr channel

ABSTRACT

Compositions and methods are provided for preventing one or more cardiac channelopathies or conditions resulting from irregularities or alterations in cardiac patterns, or both, in a human or animal subject comprising: one or more pharmacologically active agents that causes at least one of IKr channel inhibition or QT prolongation by inhibiting the activity of an ether-a-go-go-related gene (hERG); and one or more liposomes, wherein the liposomes are empty liposomes and administered prior to, concomitantly, or after administration of the pharmacologically active agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/917,426, filed Dec. 18, 2013, and U.S. Provisional ApplicationSer. No. 61/977,417, filed Apr. 9, 2014, and is a continuation-in-partapplication of U.S. patent application Ser. No. 14/268,376 filed May 2,2014, which is a continuation patent application of U.S. patentapplication Ser. No. 13/487,233, filed Jun. 3, 2012, now U.S. Pat. No.8,753,674 issued on Jun. 17,2014, which is a non-provisional applicationof U.S. Provisional Application Ser. No. 61/493,257 filed Jun. 3, 2011,the entire contents of each are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to pharmacology and cardiology,and more particularly to liposomal based compositions and methods totherapeutically alter a genetic, drug-induced inhibition of the cardiacI_(Kr) channel.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

REFERENCE TO A SEQUENCE LISTING

None.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with compositions and methods for controlling the durationof repolarization of the cardiac ventricle QT in a subject comprisingadministering to subject in need thereof of a modification of orfunctional interference with a therapeutic agent, or congenital defect,which if unmodified, can induce prolongation of repolarization in theheart myocyte action potential, torsade de points, and the long QTsyndrome. The present invention comprises of either binding a QTprolonging drug with a liposome prior to parenteral (intravenous orsubcutaneous) administration, or administration of an empty liposomeprior to or concomitantly with therapeutic agents known to have a highrisk of QT prolongation, or immediately following an envenomation.

The beating of the heart is due to precisely controlled regularly spacedwaves of myocardial excitation and contraction. The electrical currentsduring ion-based depolarization and repolarization can be measured byelectrical leads placed on the body in specific locations (theelectrocardiogram) which measure electrical waves. The P-wave representsa wave of depolarization in the atrium. When the entire atria becomesdepolarized, the wave returns to zero. After 0.1 seconds the ventricleis entirely depolarized resulting in the QRS complex. The three peaksare due to the way the current spreads in the ventricles. This isfollowed by the T-wave or repolarization of the ventricle. The QTinterval measured from the beginning of the QRS complex to the end ofthe T-wave on the standard ECG represents the duration till thecompletion of the repolarization phase of the cardiac myocyte (or thedepolarization and repolarization of the ventricle). The duration ofthis interval can vary due to genetic variation, cardiac disease,electrolyte balance, envenomation, and drugs. Prolongation of the QTinterval, can result in ventricular arrhythmias, and sudden death.

Drug induced long QTc Syndrome (LQTS) i.e., a prolongation of the actionpotential duration is a common cause of governmental mandated drugwithdrawal. QTc prolongation is an unpredictable risk factor forTorsades de Pointes (TdP), a polymorphic ventricular tachycardia leadingto ventricular fibrillation. Drug induced LQTS comprises about 3% of allprescriptions which when followed by TdP may constitute a lethal adversereaction. Patients taking one or more than one QTc-prolonging drugconcomitantly, have an enhanced risk of TdP. While the overalloccurrence of TdP is statistically rare but clinically significant forthe affected individual, assay for this drug effect is a mandatoryrequirement prior to allowing a drug to enter clinical trials.

Common structurally diverse drugs block the human ether-a-g-go-relatedgene (KCNH2 or hERG) coded K⁺ channel and the cardiac delayed-rectifierpotassium current I_(K) (KV11.1) resulting in acquired LQTS.Drug-associated increased risk of LQTS is a major drug developmenthurdle, and many drugs have been withdrawn during pre-clinicaldevelopment, or assigned black box warnings following approval, orwithdrawn from the market. Autosomal recessive or dominant LQTS basedupon 500 possible mutations in 10 different genes coding for thepotassium channel has an incidence of 1:3000 or about 100,000 persons inthe US. Prolonged QT intervals or risk of LQTS occur in 2.5% of theasymptomatic US population. This syndrome when expressed can lead tosevere cardiac arrhythmia and sudden death in untreated patients. Theprobability of cardiac death in patients with asymptomatic congenitalLQTS who are medicated with LQTS-inducing drugs is increased.

The majority of the acquired LTQS drug withdrawals are due toobstruction of the potassium ion channels coded by the humanether-a-go-go related gene (hERG). High concentrations of hERG blockingdrugs generally induce a prolonged QTc interval and increase theprobability of TdP. Up to 10% of cases of drug-induced TdP can be due to13 major genetic mutations, 471 different mutations, and 124polymorphisms (Chig, C 2006).

Systems and methods for detection of LQTS have been describedpreviously. For example U.S. Patent Publication No. 2010/0004549 (Kohlset al. 2010) discloses a system and method of detecting LQTS in apatient by comparing a collected set of ECG data from the patient to aplurality of databases of collected ECG data. The plurality of databaseswill include a database containing previous ECGs from the patient, aknown acquired LQTS characteristics database, and a known genetic LQTScharacteristics database. Comparing the patients ECG to these databaseswill facilitate the detection of such occurrences as changes in QTinterval from success of ECGs, changes in T-wave morphology, changes inU-wave morphology and can match known genetic patterns of LQTS. Thesystem and method is sensitive to patient gender and ethnicity, as thesefactors have been shown to effect LQTS, and is furthermore capable ofmatching a QT duration to a database of drug effects. The system andmethod is also easily integrated into current ECG management systems andstorage devices.

A system and method for the diagnosis and treatment of LQTS is describedin U.S. Patent Publication No. 2008/0255464 (Michael, 2008). The Michaelinvention includes a system for diagnosing Long QT Syndrome (LQTS)derives a QT/QS2 ratio from an electrical systole (QT) and a mechanicalsystole (QS2) to detect a prolonged QT interval in a patient's cardiaccycle. A processor acquires the systoles from a microphone and chestelectrodes, calculates the QT/QS2 ratio, and outputs the result to adisplay. The processor may compare the QT/QS2 ratio to a threshold valuestored in memory for diagnosing LQTS in the patient. A user interfaceprovides for programming, set-up, and customizing the display. A modeselector allows the system to operate alternatively as aphonocardiograph, a 12 lead electrocardiograph, or a machine fordiagnosing LQTS. A related method for diagnosing cardiac disorders suchas LQTS includes measuring QT and QS2 during a same cardiac cycle,calculating a QT/QS2 ratio, and comparing the result to a thresholdvalue derived from empirical data. The method may include measuringsystoles both at rest and during exercise, and may be used for drugefficacy, dosage optimization, and acquired LQTS causality tests.

A method for the treatment of cardiac arrhythmias is provided in U.S.Patent Publication No. 2007/0048284 (Donahue and Marban, 2007). Themethod includes administering an amount of at least one polynucleotidethat modulates an electrical property of the heart. The polynucleotidesof the invention may also be used with a microdelivery vehicle such ascationic liposomes and adenoviral vectors.

Methods, compositions, dosing regimes, and routes of administration forthe treatment or prevention of arrhythmias have been described by Fedidaet al. (2010) in U.S. Patent Publication No. 2001/00120890. In theFedida invention, early after depolarizations and prolongation of QTinterval may be reduced or eliminated by administering ion channelmodulating compounds to a subject in need thereof. The ion channelmodulating compounds may be cycloalkylamine ether compounds,particularly cyclohexylamine ether compounds. Also described arecompositions of ion channel modulating compounds and drugs which induceearly after depolarizations, prolongation of QT interval and/or Torsadesde Pointes. The Fedida invention also discloses antioxidants which maybe provided in combination with the ion channel modulating compounds,non-limiting examples of the antioxidants include vitamin C, vitamin E,beta-carotene, lutein, lycopene, vitamin B2, coenzyme Q10, cysteine aswell as herbs, such as bilberry, turmeric (curcumin), grape seed or pinebark extracts, and ginkgo.

SUMMARY OF THE INVENTION

Crizotinib (Xalkori®) and nilotinib (Tasigna®) are tyrosine kinaseinhibitors approved for the treatment of non-small cell lung cancer andchronic myeloid leukemia, respectively. Both have been shown to resultin QT prolongation in humans and animals. Liposomes have been shown toameliorate drug-induced effects on the IKr (KV11.1) channel, coded bythe human ether-a-go-go-related gene (hERG). This study was done todetermine if liposomes would also decrease the effect of crizotinib andnilotinib on the IKr channel. Crizotinib and nilotinib were tested in astandard in vitro IKr assay using human embryonic kidney (HEK) 293 cellsstably transfected with the hERG. Dose-responses were determined and 50%inhibitory concentrations (IC₅₀s) were calculated. When the HEK 293cells were treated with crizotinib and nilotinib that were mixed withliposomes, there was a significant decrease in the IKr channelinhibitory effects of these two drugs. The use of liposomal encapsulatedQT-prolongation agents, or just mixing these drugs with liposomes, e.g.,empty liposomes, was found to decrease their cardiac liability.

In one embodiment, the present invention includes a composition forpreventing one or more cardiac channelopathies or conditions resultingfrom irregularities or alterations in cardiac patterns, or both, in ahuman or animal subject comprising: one or more pharmacologically activeagents that causes at least one of IKr channel inhibition or QTprolongation by inhibiting the activity of an ether-a-go-go-related gene(hERG); and one or more liposomes, wherein the liposomes are emptyliposomes and administered prior to, concomitantly, or afteradministration of the pharmacologically active agent. In one aspect, thecardiac channelopathy or the condition resulting from the irregularityor alteration in the cardiac pattern is inhibition of an ion channelresponsible for the delayed-rectifier K+ current in the heart,polymorphic ventricular tachycardia, prolongation of the QTc, LQT2,LQTS, or torsades de pointes. In another aspect, the composition is usedfor the treatment or prevention of prolongation of the IKr channelinhibition or QT prolongation induced by administration of one or moredrugs used in the treatment of cardiac or non-cardiac related diseases.In another aspect, the one or more active agents is selected from atleast one of crizotinib, nilotinib, terfenadine, astemizole,gripafloxacin, terodilene, droperidole, lidoflazine, levomethadyl,sertindoyle or cisapride. In another aspect, the one or more activeagents is selected from at least one of: Aloxi; Amiodarone; Arsenictrioxide; Astemizole; Bepridil; Chloroquine; Chlorpheniramine;Chlorpromazine (Thorazine); Cisapride; Celaxa; Citalopram;Clarithromycin; Erythromycin; Curcumin; Disopyramide; Dofetilide;Domperidone; Doxorubicin; Dronedarone; Droperidol; Grepafloxacin;Haldol; Haloperidol; Halofantrine; Ibutilide; Levomethadyl; Lidoflazine;Loratidine; Lovostatin; Mesoridazone; Methadone; Methanesulphonanilide;Moxifloxacin; Palonasitron; Pentamadine; Pimozide; Prenylamine;Probucol; Procainamide; Propafenone; Pyrilamine; Quinidine; Terfenidine;Sertindole; Sotalol; Sparfloxacin; Thioridazine; or Vandetanib. Inanother aspect, the composition is adapted for enteral, parenteral,intravenous, intraperitoneal, or oral administration. In one aspect, thecomposition consists essentially of the therapeutically effective amountof a composition comprising: one or more pharmacologically active agentsthat cause cardiac channelopathies or conditions resulting fromirregularities or alterations in cardiac patterns and the emptyliposomes, wherein the amount of empty liposomes is sufficient to reducethe cardiac channelopathies or conditions resulting from irregularitiesor alterations in cardiac patterns caused by the active agent. Inanother aspect, the active agent and the liposomes may be bound orconjugated together. In another aspect, the liposomes comprise anionic,cationic, or neutral liposomes. In another aspect, the liposomescomprises a lipid or a phospholipid wall, wherein the lipids or thephospholipids are selected from the group consisting ofphosphatidylcholine (lecithin), lysolecithin,lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol,sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine,and dipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine,hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid,fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, anddiacylglycerolsuccinate. In another aspect, the composition furthercomprises a pharmaceutically acceptable dispersion medium, solvent, orvehicle, wherein the active agent, the liposome or both are dissolved,dispersed, or suspended in the medium, the solvent, or the vehicle. Inanother aspect, the liposomes comprise DMPC(1,2-dimyristoil-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]). In anotheraspect, the liposomes comprise a 9.7:1 ratio of DMPC(1,2-dimyristoil-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3 -phospho-rac-[1-glycerol]).

In one embodiment, the present invention includes a composition forpreventing or treating one or more adverse reactions arising fromadministration of a therapeutically active agent or a drug in a humanthat causes at least one of IKr channel inhibition or QT prolongation byinhibiting the activity of an ether-a-go-go-related gene (hERG)comprising: one or more pharmacologically active agents that causes atleast one of IKr channel inhibition or QT prolongation and one or moreliposomes, wherein the liposomes are empty liposomes and administeredprior to, concomitantly, or after administration of the therapeuticallyactive agent or the drug and the liposomes are provided in an amountsufficient to reduce or eliminate the IKr channelopathy or QTprolongation. In one aspect, the therapeutically active agent or a drugis used in a prevention or a treatment of one or more cardiac ornon-cardiac diseases in the human or animal subject. In another aspect,the cardiac channelopathy or the condition resulting from theirregularity or alteration in the cardiac pattern is inhibition of anion channel responsible for the delayed-rectifier K+ current in theheart, polymorphic ventricular tachycardia, prolongation of the QTc,LQT2, LQTS, or torsades de pointes. In another aspect, the compositionis used for the treatment or prevention of prolongation of the IKrchannel inhibition or QT prolongation induced by administration of oneor more drugs used in the treatment of cardiac or non-cardiac relateddiseases. In another aspect, the one or more active agents is selectedfrom at least one of crizotinib, nilotinib, terfenadine, astemizole,gripafloxacin, terodilene, droperidole, lidoflazine, levomethadyl,sertindoyle or cisapride. In another aspect, the one or more activeagents is selected from at least one of: Aloxi; Amiodarone; Arsenictrioxide; Astemizole; Bepridil; Chloroquine; Chlorpheniramine;Chlorpromazine (Thorazine); Cisapride; Celaxa; Citalopram;Clarithromycin; Erythromycin; Curcumin; Disopyramide; Dofetilide;Domperidone; Doxorubicin; Dronedarone; Droperidol; Grepafloxacin;Haldol; Haloperidol; Halofantrine; Ibutilide; Levomethadyl; Lidoflazine;Loratidine; Lovostatin; Mesoridazone; Methadone; Methanesulphonanilide;Moxifloxacin; Palonasitron; Pentamadine; Pimozide; Prenylamine;Probucol; Procainamide; Propafenone; Pyrilamine; Quinidine; Terfenidine;Sertindole; Sotalol; Sparfloxacin; Thioridazine; or Vandetanib. Inanother aspect, the composition is adapted for enteral, parenteral,intravenous, intraperitoneal, or oral administration. In another aspect,the active agent and the liposomes may be bound or conjugated together.In another aspect, the liposomes comprise anionic, cationic, or neutralliposomes. In another aspect, the liposomes comprises a lipid or aphospholipid wall, wherein the lipids or the phospholipids are selectedfrom the group consisting of phosphatidylcholine (lecithin),lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine,phosphatidylinositol, sphingomyelin, phosphatidylethanolamine(cephalin), cardiolipin, phosphatidic acid, cerebrosides,dicetylphosphate, phosphatidylcholine, anddipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine,hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid,fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, anddiacylglycerolsuccinate. In another aspect, the liposomes are sphericalliposomes with a diameter ranging from 10 nm-200 nm. In another aspect,the liposomes comprise DMPC(1,2-dimyristoil-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]). In anotheraspect, the liposomes comprise a 9.7:1 ratio of DMPC(1,2-dimyristoil-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3 -phospho-rac-[1-glycerol]).

In another embodiment, the present invention includes a method forpreventing or treating one or more cardiac channelopathies,irregularities or alterations in cardiac patterns, or both in a human oranimal subject comprising the steps of: identifying the human or animalsubject in need of prevention or treatment of the one or more cardiacchannelopathies, irregularities or alterations in cardiac patterns, orboth; and administering to the human or animal subject a therapeuticallyeffective amount of a composition comprising: one or morepharmacologically active agents that causes at least one of IKr channelinhibition or QT prolongation by inhibiting the activity of anether-a-go-go-related gene (hERG); one or more liposomes, wherein theliposomes are empty liposomes and administered prior to, concomitantly,or after administration of the pharmacologically active agent in anamount sufficient to prevent or treat one or more cardiacchannelopathies, irregularities or alterations in cardiac patterns; andan optional pharmaceutically acceptable dispersion medium, solvent, orvehicle, wherein the active agent, the liposome or both are dissolved,dispersed, or suspended in the medium, the solvent, or the vehicle. Inone aspect, the composition consists essentially of the therapeuticallyeffective amount of a composition comprising: one or morepharmacologically active agents that causes at least one of IKr channelinhibition or QT prolongation by inhibiting the activity of anether-a-go-go-related gene (hERG) and the empty liposomes, wherein theamount of empty liposomes is sufficient to reduce the IKr channelinhibition or QT prolongation. In one aspect, the cardiac channelopathyor the condition resulting from the irregularity or alteration in thecardiac pattern is inhibition of an ion channel responsible for thedelayed-rectifier K+ current in the heart, polymorphic ventriculartachycardia, prolongation of the QTc, LQT2, LQTS, or torsades depointes. In another aspect, the one or more active agents is selectedfrom at least one of crizotinib, nilotinib, terfenadine, astemizole,gripafloxacin, terodilene, droperidole, lidoflazine, levomethadyl,sertindoyle or cisapride. In another aspect, the liposomes comprise DMPC(1,2-dimyristoil-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]). In anotheraspect, the liposomes comprise a 9.7:1 ratio of DMPC(1,2-dimyristoil-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]). In anotheraspect, the one or more active agents is selected from at least one of:Aloxi; Amiodarone; Arsenic trioxide; Astemizole; Bepridil; Chloroquine;Chlorpheniramine; Chlorpromazine (Thorazine); Cisapride; Celaxa;Citalopram; Clarithromycin; Erythromycin; Curcumin; Disopyramide;Dofetilide; Domperidone; Doxorubicin; Dronedarone; Droperidol;Grepafloxacin; Haldol; Haloperidol; Halofantrine; Ibutilide;Levomethadyl; Lidoflazine; Loratidine; Lovostatin; Mesoridazone;Methadone; Methanesulphonanilide; Moxifloxacin; Palonasitron;Pentamadine; Pimozide; Prenylamine; Probucol; Procainamide; Propafenone;Pyrilamine; Quinidine; Terfenidine; Sertindole; Sotalol; Sparfloxacin;Thioridazine; or Vandetanib.

In yet another embodiment, the present invention includes a method forpreventing or treating one or more adverse reactions arising fromadministration of a therapeutically active agent or a drug in a human oranimal subject comprising the steps of: identifying the human or animalsubject in need of prevention or treatment of the one or more adversereactions arising from the administration of the therapeutically activeagent or the drug that causes at least one of IKr channel inhibition orQT prolongation by inhibiting the activity of an ether-a-go-go-relatedgene (hERG); and administering to the human or animal subject atherapeutically effective amount of a composition comprising one or moreliposomes, wherein the liposomes are empty liposomes and administeredprior to, concomitantly, or after administration of the therapeuticallyactive agent or the drug or are liposomes loaded with thetherapeutically active agent or the drug; and measuring the effect ofthe combination of the liposomes and the therapeutically active agent orthe drug on the drug-induced channelopathy, wherein the compositionreduces or eliminated the channelopathy induced by the therapeuticallyactive agent or the drug. In one aspect, the one or more active agentsor drugs is selected from at least one of: Aloxi; Amiodarone; Arsenictrioxide; Astemizole; Bepridil; Chloroquine; Chlorpheniramine;Chlorpromazine (Thorazine); Cisapride; Celaxa; Citalopram;Clarithromycin; Erythromycin; Curcumin; Disopyramide; Dofetilide;Domperidone; Doxorubicin; Dronedarone; Droperidol; Grepafloxacin;Haldol; Haloperidol; Halofantrine; Ibutilide; Levomethadyl; Lidoflazine;Loratidine; Lovostatin; Mesoridazone; Methadone; Methanesulphonanilide;Moxifloxacin; Palonasitron; Pentamadine; Pimozide; Prenylamine;Probucol; Procainamide; Propafenone; Pyrilamine; Quinidine; Terfenidine;Sertindole; Sotalol; Sparfloxacin; Thioridazine; or Vandetanib.

In another embodiment, the present invention includes a method forpreventing or treating at least one of IKr channel inhibition or QTprolongation arising from administration of crizotinib, nilotinib, orany other active agent that causes a drug-induced channelopathy in ahuman or animal subject comprising the steps of: identifying the humanor animal subject in need of prevention or treatment at least one of IKrchannel inhibition or QT prolongation that results from theadministration of crizotinib, nilotinib, or any other active agent thatcauses a drug-induced channelopathy; and administering to the human oranimal subject a therapeutically effective amount of a compositioncomprising one or more liposomes, wherein the liposomes are emptyliposomes and administered prior to, concomitantly, or afteradministration of the crizotinib, nilotinib, or any other active agentthat causes a drug-induced channelopathy, wherein the compositionreduces or eliminated the channelopathy induced by the therapeuticallyactive agent or the drug. In one aspect, the active agent has previouslyfailed a clinical trial due to drug-induced IKr channel inhibition or QTprolongation. In another aspect, the method further comprises the stepof identifying a drug in a clinical trial that failed or has limitedclinical use due to drug-induced IKr channel inhibition or QTprolongation side-effects. In one aspect, the one or more active agentsis selected from at least one of: Aloxi; Amiodarone; Arsenic trioxide;Astemizole; Bepridil; Chloroquine; Chlorpheniramine; Chlorpromazine(Thorazine); Cisapride; Celaxa; Citalopram; Clarithromycin;Erythromycin; Curcumin; Disopyramide; Dofetilide; Domperidone;Doxorubicin; Dronedarone; Droperidol; Grepafloxacin; Haldol;Haloperidol; Halofantrine; Ibutilide; Levomethadyl; Lidoflazine;Loratidine; Lovostatin; Mesoridazone; Methadone; Methanesulphonanilide;Moxifloxacin; Palonasitron; Pentamadine; Pimozide; Prenylamine;Probucol; Procainamide; Propafenone; Pyrilamine; Quinidine; Terfenidine;Sertindole; Sotalol; Sparfloxacin; Thioridazine; or Vandetanib.

In another embodiment, the present invention includes a method ofevaluating a candidate drug that reduces a channelopathy caused by apharmacologically active agent, the method comprising: (a) administeringa candidate drug to a first subset of the patients, and a placebo to asecond subset of the patients, wherein the composition is provided inconjunction with the pharmacologically active agent that causes at leastone of I_(Kr) channel inhibition or QT prolongation and one or moreliposomes, wherein the liposomes are empty liposomes and administeredprior to, concomitantly, or after administration of the therapeuticallyactive agent or the drug; (b) measuring the channelopathy from asuspected of having a drug-induced channelopathy from a set of patients;(c) repeating step (a) after the administration of the candidate drug orthe placebo; and (d) determining if the composition reduces thedrug-induced channelopapthy that is statistically significant ascompared to any reduction occurring in the second subset of patients,wherein a statistically significant reduction indicates that thecandidate drug is useful in treating said disease state. In one aspect,the drug has previously failed a clinical trial due to drug-induced IKrchannel inhibition or QT prolongation. In another aspect, the one ormore active agents is selected from at least one of: Aloxi; Amiodarone;Arsenic trioxide; Astemizole; Bepridil; Chloroquine; Chlorpheniramine;Chlorpromazine (Thorazine); Cisapride; Celaxa; Citalopram;Clarithromycin; Erythromycin; Curcumin; Disopyramide; Dofetilide;Domperidone; Doxorubicin; Dronedarone; Droperidol; Grepafloxacin;Haldol; Haloperidol; Halofantrine; Ibutilide; Levomethadyl; Lidoflazine;Loratidine; Lovostatin; Mesoridazone; Methadone; Methanesulphonanilide;Moxifloxacin; Palonasitron; Pentamadine; Pimozide; Prenylamine;Probucol; Procainamide; Propafenone; Pyrilamine; Quinidine; Terfenidine;Sertindole; Sotalol; Sparfloxacin; Thioridazine; or Vandetanib. In oneaspect, the method is conducted in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 is a graph that shows the effect of terfenadine on hERG currentdensity from transfected HEK 293 cells at 20 mV;

FIG. 2 is a graph that shows the current-voltage (I-V) relationship ofhERG current amplitude from transfected HEK 293 cells exposed toterfenadine;

FIG. 3 is a graph that shows the effect of terfenadine on hERG currentdensity from transfected HEK 293 cells at 20 mV;

FIG. 4 is a graph that shows the I-V relationship of hERG currentamplitude from transfected HEK 293 cells exposed to terfenadine;

FIG. 5 is a graph that shows the effect of E-4031 on hERG currentdensity from transfected HEK 293 cells at 20 mV;

FIG. 6 is a graph that shows the effect of curcumin on hERG currentdensity from transfected HEK 293 cells at 20 mV;

FIG. 7 is a graph that shows the I-V relationship of hERG currentamplitude from transfected HEK 293 cells exposed to curcumin;

FIG. 8 is a graph that shows the effect of curcumin (as liposomalcurcumin) on hERG current density from transfected HEK 293 cells at 20mV;

FIG. 9 is a graph that shows the I-V relationship of hERG currentamplitude from transfected HEK 293 cells exposed to Curcumin (asliposomal curcumin);

FIG. 10 is a graph that shows the effect of Curcumin(Liposomes+Curcumin) on hERG current density from transfected HEK 293cells at 20 mV;

FIG. 11 is a graph that shows the I-V relationship of hERG currentamplitude from transfected HEK 293 cells exposed to Curcumin(Liposomes+Curcumin);

FIG. 12 is a graph that shows the effect of liposomes on hERG currentdensity from transfected HEK 293 cells at 20 mV;

FIG. 13 is a graph that shows the I-V relationship of hERG currentamplitude from transfected HEK 293 cells exposed to liposomes;

FIG. 14 is a graph that shows the of liposomes+E-4031 on hERG currentdensity from transfected HEK 293 cells at 20 mV;

FIG. 15 is a graph that shows the I-V relationship of hERG currentamplitude from transfected HEK 293 cells exposed to Liposomes+E-4031;

FIG. 16 is a graph that shows the effect of liposomes+terfenadine onhERG current density from transfected HEK 293 cells at 20 mV;

FIG. 17 is a graph that shows the I-V relationship of hERG currentamplitude from transfected HEK 293 cells exposed toliposomes+terfenadine;

FIGS. 18A and 18B is a graph that shows the IKr tail current densityaverages and voltage dependency obtained by measuring the IKr tail peakamplitude and in the presence of crizotinib, liposomes alone, andcrizotinib plus liposomes;

FIGS. 19A and 19B is a graph that shows the IKr tail current densityaverages and voltage dependency obtained by measuring the IKr tail peakamplitude and in the presence of nilotinib, liposomes alone, andnilotinib plus liposomes;

FIG. 20 is a graph that shows the in vivo effect of Crizotinib andLiposomes+Crizotinib on RR interval (ms) of rabbit heart;

FIG. 21 is a graph that shows the in vivo effect of Crizotinib andLiposomes+Crizotinib on PR interval (ms) of rabbit heart;

FIG. 22 is a graph that shows the in vivo effect of Crizotinib andLiposomes+Crizotinib on QRS interval (ms) of rabbit heart;

FIG. 23 is a graph that shows the in vivo effect of Crizotinib andLiposomes+Crizotinib on QT interval (ms) of rabbit heart;

FIG. 24 is a graph that shows the in vivo effect of Crizotinib andLiposomes+Crizotinib on QTc Van der Water intervals of rabbit heart;

FIG. 25 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on RR interval (ms) of rabbit heart;

FIG. 26 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on PR interval (ms) of rabbit heart;

FIG. 27 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on QRS interval (ms) of rabbit heart;

FIG. 28 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on QT interval (ms) of rabbit heart;

FIG. 29 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on QTc Van der Water intervals of rabbit heart;and

FIGS. 30A and 30B are graphs that show QTc prolongation in rabbitstreated with crizotinib, nilotinib, crizotinib plus liposomes, andnilotinib plus liposomes. Animals were given an IV loading dose over a10 minute period, followed by a maintenance dose over a 15 minuteperiod. Liposomes were dosed IV 5 minutes before the loading dose of thedrugs. The loading and maintenance doses for crizotinib were 1, 2 and 3mg/kg, and 0.4, 0.8 and 1.2 mg/kg, respectively (FIG. 30A). The dosesfor nilotinib were 2, 4 and 5.5 mg/kg, and 0.14, 0.28 and 0.39 mg/kg,respectively (FIG. 30B). The doses of liposomes were 9-fold higher thanthe doses of drug, on a mg/kg basis. The values plotted are themean+standard error of the mean, for 3 rabbits per group. Statisticalcomparisons were done as described in FIGS. 23 and 28.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein the term “Curcumin”, “diferuloylmethane”, or“1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)” is anaturally occurring compound which is the main coloring principle foundin the rhizomes of the plant Curcuma longa (see e.g., U.S. Pat. No.5,679,864, Krackov et al.).

The term “liposome” refers to a capsule wherein the wall or membranethereof is formed of lipids, especially phospholipid, with the optionaladdition therewith of a sterol, especially cholesterol.

As used herein, the term “in vivo” refers to being inside the body. Theterm “in vitro” used as used in the present application is to beunderstood as indicating an operation carried out in a non-livingsystem.

As used herein, the term “receptor” includes, for example, moleculesthat reside on the surface of cells and mediate activation of the cellsby activating ligands, but also is used generically to mean any moleculethat binds specifically to a counterpart. One member of a specificbinding pair would arbitrarily be called a “receptor” and the other a“ligand.” No particular physiological function need be associated withthis specific binding. Thus, for example, a “receptor” might includeantibodies, immunologically reactive portions of antibodies, moleculesthat are designed to complement other molecules, and so forth. Indeed,in the context of the present invention, the distinction between“receptor” and “ligand” is entirely irrelevant; the invention concernspairs of molecules, which specifically bind each other with greateraffinity than either binds other molecules. However, for ease ofexplanation, the invention method will be discussed in terms of targetreceptor (again, simply a molecule for which a counterpart is soughtthat will react or bind with it) and “ligand” simply represents thatcounterpart.

As used herein, the term “treatment” refers to the treatment of theconditions mentioned herein, particularly in a patient who demonstratessymptoms of the disease or disorder.

As used herein, the term “treating” refers to any administration of acompound of the present invention and includes (i) inhibiting thedisease in an animal that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., arresting further development ofthe pathology and/or symptomatology); or (ii) ameliorating the diseasein an animal that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., reversing the pathology and/orsymptomatology). The term “controlling” includes preventing treating,eradicating, ameliorating or otherwise reducing the severity of thecondition being controlled.

The terms “effective amount” or “therapeutically effective amount”described herein means the amount of the subject compound that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician.

The terms “administration of” or “administering a” compound as usedherein should be understood to mean providing a compound of theinvention to the individual in need of treatment in a form that can beintroduced into that individual's body in a therapeutically useful formand therapeutically useful amount, including, but not limited to: oraldosage forms, such as tablets, capsules, syrups, suspensions, and thelike; injectable dosage forms, such as IV, IM, or IP, and the like;transdermal dosage forms, including creams, jellies, powders, orpatches; buccal dosage forms; inhalation powders, sprays, suspensions,and the like; and rectal suppositories.

As used herein the term “intravenous administration” includes injectionand other modes of intravenous administration.

The term “pharmaceutically acceptable” as used herein to describe acarrier, diluent or excipient must be compatible with the otheringredients of the formulation and not deleterious to the recipientthereof

The term “hERG” as used herein refers to the human Ether-a-go-go-RelatedGene, which is encoded by the KCNH2 gene (see www.genecards.com). TheKCNH2 gene encodes a protein also known as Kvl 1.1, which is the alphasubunit of a potassium ion channel that contributes to the electricalactivity of the heart that coordinates the heart's beating.Specifically, the hERG-containing channel mediates the repolarizing IKrcurrent in the cardiac action potential.

Inhibition of the ion channel's ability to conduct electrical currentacross the cell membrane results in a potentially fatal disorder calledlong QT syndrome. A number of clinically successful drugs in the markethave been found to inhibit hERG, thus creating a risk of sudden death.This drug-induced side-effect has caused hERG inhibition a key target tobe avoided during drug development.

The term “I_(Kr) channel” as used herein refers to the ‘rapid’ delayedrectifier current Oa that conducts potassium (K⁻) ions out of the musclecells of the heart (cardiac myocytes). This current is critical incorrectly timing the return to the resting state (repolarization) of thecell membrane during the cardiac action potential. Often, the terms“hERG channels” and I_(Kr) are used interchangeably, although hERG formspart of naturally occurring channels in the body, which are morecommonly referred to by the name of the electrical current that has beenmeasured in that cell type, which in the case of the heart is I_(Kr).

EXAMPLE 1 Compositions and Methods for Controlling the Duration ofRepolarization of the Cardiac Ventricle QT Interval

Compositions and methods for controlling the duration of repolarizationof the cardiac ventricle QT interval are disclosed herein. The method ofthe present invention comprises comprising administering to subject inneed thereof of a modification of or a functional interference with atherapeutic agent, or a congenital defect which if unmodified can induceprolongation of repolarization in the heart myocyte action potential,torsade de points, and the long QT syndrome. The present inventioncomprises of either binding a QT prolonging drug with a liposome priorto parenteral (intravenous or subcutaneous) administration, or emptyliposomal administration prior to or concomitantly with one or moretherapeutic agents known to have a high risk of QT prolongation, orimmediately following an envenomation. The findings of the presentinvention indicate that the adverse effect of curcumin and other QTprolonging drugs is abrogated with liposomal curcumin, and with vortexedmixtures of empty liposomes in a dose dependent manner.

Ion channels are pore-forming integral membrane proteins that establishand control the electrochemical gradient (the action potential) acrossthe plasma membrane, and intracellular organelles of cells by modulatingion. The channels are assembled as a circular arrangement of proteinspacked around a water-filled pore. The ions passage through the channelin single file, which may be open or closed by chemical, electricalsignals, temperature, or mechanical force. Ion channel dysfunction maybe associated with mutations in the genes coding these channels or withdrugs interfering with ion flow. Dysfunction in cardiac electrolytepotassium, calcium, and sodium channels in the cardiac myocyte membraneinduces defects in electrical currents, and the normal action potentialwhich are necessary for coordinated myocyte contraction and maintenanceof normal blood circulation resulting in clinical cardiac symptoms. Thecentral roles of the 40 members, and 12 subfamilies of voltage gatedpotassium channel's (Kv) role are to repolarize the cell membranefollowing action potentials. The flux of potassium ions in the cardiacmyocyte K⁺ channels modulates electrolytic currents, levels ofdepolarization and repolarization. Congenital and/or drug-inducedchannel defects are associated with morbidity and mortality in otherwiseasymptomatic individuals. The channel proper coded by the gene KCNH2 orhERG (human ether-a-go-go-related gene) contains proteins designated asKv11.1 and the Lv11.1 α-subunit of the rapidly activating rectifier K⁺current I_(Kr). This cell membrane channel mediates the “rapid” delayedrectifier current I_(Kr) by conducting K⁺ ions out of the cardiacmyocytes and is a critical mechanism to allow the cardiac potential toreturn to the resting state (repolarization).

Even though the hERG channel pore-domain lacks a known three-dimensionalstructure, insight into its putative structure has been gained fromsite-directed mutagenesis data (Stansfeld P J, 2007). Within the hERGchannel pore cavity, ion flux and currents can be modified dependingupon the open or closed states, and by drug interactions at key highaffinity drug binding sites. These sites are the aromatic amino-acidresidues (Y652 and F656) on the inner helices of the pore. The mostimportant currents mediated by drugs, the sensitive delayed, I_(Kr)(rapid) current which repolarizes the myocardial cells and the I_(Ks)(slow) rectifier currents are exhibited on the standardelectrocardiogram (ECG) as the QT interval which when corrected forheart rate this is conventionally defined as QTc.

Congenital defects in ion channels first described by Jervell A, (1957),alter the balance of currents determining repolarization of the actionpotential and predispose to LQTS arrhythmias.and sudden cardiac death.Mutations have been identified giving rise to subtypes of congenitalLQTS, familial arrhythmogenic syndromes characterized by abnormal ionchannel function, delayed repolarization, prolonged QT interval on theelectrocardiogram and a life-threatening polymorphic ventriculartachycardia known as torsade de points. Different mutations in the hERGgene and its coded proteins translate to defects in channel function anda number of clinical syndromes. Type 2 congenital long-QT syndrome(LQT2) results from A614V missense mutations in the KCNH gene and ischaracterized by four classes of loss of Kv11.1 protein and consequentchannel dysfunction. These abnormal Kv11.1 channels include (Class 1), adominant—intracellular trafficking-deficient ion channel protein:usually due to missense mutations, (Class 2), a correctible phenotypewhen cells are incubated for 24 hours at 27° temperature, or withexposure to the drugs E-4031 (Zhou Z 1999 (Class 3)), channel gating,and (Class 4) permeation)(Anderson C.L, 2006). Blockade by any of theseand particularly the “rapid” current prolongs the action potential andmanifests on the ECG as a prolonged QT interval and emergence of other Tor U wave abnormalities. Under such circumstances, activation of aninward depolarization current induces increased dispersion ofrepolarization. The latter results in a heterogeneous recovery ofexcitability, and induction of torsades de points (TdP) an earlypremature ventricular contraction (PVC). (R-On-T). This is whereventricular depolarization i.e, the R-wave occurs simultaneously withthe relative refractory period at the end of repolarization (latter halfof the T-wave) and initiates pathologic T-U waves and torsades.Sustained TdP leads to a zone of functional refractoriness in themyocardium, and cardiac arryhthmias. The ECG reading in torsadesexhibits a rapid polymorphic ventricular tachycardia with acharacteristic twist of the QRS complex around the isoelectric baseline.This is characterized by a rotation of the heart's electrical axis by asmuch as 180°, long and short RR-intervals, and clinically this leads toa fall in arterial blood pressure, syncope, degeneration intoventricular fibrillation and sudden death.

On the ECG, retardation of the I_(Kr) current interval is synonymouswith QT prolongation when greater than 440 ms in men and 460 ms inwomen. Pharmacological inhibition of hERG K⁺ channels by structurallyand therapeutically diverse drugs translates to the clinical acquiredform of the long QT syndrome (LQTS). While QT prolonging drugs representtwo to three percent of the total prescriptions in the developed worldthe reported incidence of QT prolongation and dosage variessignificantly within different drug classes. The latter include Class 1Aand Class III antiarrhythmics, antihistamines, antimicrobials,antipsychotics, tricyclic antidepressants, prokinetics, andanti-anginals. Recently, curcuminoids were reported to block humancardiac K⁺ channels. (Moha ou Maati H, 2008).

Increased incidence of QT prolongation may also occur in the presence ofhypomagnesemia, hypokalemia, hypocalcalcemia, hypoxia, acidosis, heartfailure, left ventricular hypertrophy, slow heart rate, female gender,hypothermia, and subarachnoid hemorrhage. The severity of arrhythmia ata given QT interval, and development of TdP varies from drug to drug andpatient to patient and may not be linearly related to the dose or plasmaconcentration of a specific drug. However, antiarrhythmic cardiac drugsaffecting the potassium (K⁺) efflux (Class III) and non-cardiac drugs:that significantly alter repolarization, as measured by prolongation ofthe QT interval predispose the patient to torsades. Additional factorsassociated with an increased tendency toward TdP include familial longQT syndrome (LQTS). The most common causes of familial LQTS aremutations in genes.

KCNQ1 codes for KvLTQ1, the alpha subunit of the slow delayed potassiumrectifier potassium channel is highly expressed in the heart. Thecurrent through the heteromeric channel when interacting with the minKbeta subunit is known as I_(Ks). When missense mutated it reduces theamount of repolarizing current needed to terminate the action potential.These LTQ1 mutations represent 35% of all cases, and are the leastsevere, usually causing syncope.

KCNH2 or the hERG gene when mutated represents 30% of all genetic cases,and is the subunit of the rapid delayed rectifier potassium channelhERG+MiRP 1. Current through this channel known as I_(Kr) is responsiblefor termination of the action potential and the length of the QTinterval. When reduced it leads to LQT2. The rapid current is not onlythe most drug sensitive, but also is associated with the pro-arrhythmiceffect in His-Purkinje cells and M cells in the mid-ventricularmyocardium. Drug induced LQTs occurs with anti-arrhythmic drugs,antihistamines, anti-psychotic and other drugs. The combination ofgenetic LQTS and LQTS-inducing drugs increase susceptibility to lethalside effects. Most drugs causing LKTS block the I_(Kr) current via thehERG gene. This channel exhibits unintended drug binding at tyrosine 652and phenylalanine 656 which when bound block current conduction.Uncommon but lethal mutations in gene SCN5A slow inactivation of thealpha subunit of the sodium channel, prolonging Na⁺ influx and thecurrent I_(Na) during depolarization. Continued depolarizing currentthrough the channel late in the action potential induces a late burstingcurrent (LQT3).

L-type calcium channels re-open during the plateau phase of the actionpotential following LQTS as “early after depolarizations.” Theiractivity is sensitive to adrenergic stimulation and increases the riskof sudden death during adrenergic states in the presence of impairedrepolarization. In these subjects TdP can be precipitated followingexercise, or emotional surprise unrelated to drugs. There are additionaluncommon and rare mutations designated LQT4-13.

Apart from heart rate, the QT duration varies with recording andmeasurement techniques, sympatho-vagal activity, drugs, electrolytedisorders, cardiac or metabolic diseases, diurnal variation and geneticLQT2 mutations. These parameters cause the reported incidence ofdrug-induced TdP to be loosely associated with clinical studies duringdrug development, post-marketing surveillance, epidemiologic studies,and anecdotal case reports. Detection of QT prolongation duringpre-clinical drug development can lead to abandonment and precludes anyall-inclusive accounting of the actual incidence of drug related QTprolongation (Yap 2003). A number of QT-prolonging drugs have beenwithdrawn either during development or after being on the market. Theseinclude Terfenadine, Astemizole, Gripafloxacin, Terodilene, Droperidole,Lidoflazine, Levomethadyl, Sertindoyle, levomethadyl, and Cisapride.

Genetic and age related susceptibility: there are pre-dispositions toQT-prolonging drug events: this includes patients with structural heartdisease, taking hepatic C450 inhibitors, who have a geneticpredisposition, or DNA polymorphisms. Old females generally are moresusceptible than young females, while young males have increasedsusceptibility compared to elderly males.

Current Therapy for QT prolonging-drugs, and in genotypic QTsensitivity: Pharmacological therapy: first line treatment for LQTS, apotentially lethal disease with a 13% incidence of cardiac arrest andsudden death. (i) Dexrazoxane: (a piperazinedione cyclic derivative ofedetic acid). It diminishes but does not eliminate the potential foranthracycline induced cardiotoxicity associated with over 300 mg/M²epirubicin administered to patients with breast cancer. Use ofintravenous Dexrazoxane is limited to anthracyclines only, i.e. it iscontraindicated in chemotherapy regimens that do not contain ananthracycline. (ii) β-blockers: propranolol as sympathetic stimulationtherapy may decrease risk of cardiac events by 81% in LQT1, it may alsosuppress isoproteranolol augmentation of transmural dispersion ofrepolarization(TDR) and TdP, however on adequate propranolol treatment10% still develop cardiac events. In LQT2 subjects, cardiac event riskis decreased 59%, however 23% still develop cardiac events. (iii) Sodiumchannel blockers: 32% of LQT3 subjects develop cardiac events onadequate propranolol. In these subjects with low heart rates, β-blockersmay increase dispersion of repolarization and risk of TdP. LQT3 subjectswith sodium channel mutations preventing inactivation and inducingpersistent increase in late I_(Na) during phase 2 of the actionpotential, a cause of QT prolongation using, mexiletine (Shimizu W,1997) a Class IB sodium channel blocker abbreviates the QT interval byreduction of TDR. (iv) Potassium supplementation: both I_(Kr) and I_(K1)are sensitive to extracellular potassium levels. Raising plasmaconcentration by 1.5 mEq/L above baseline can reduce the QTc interval by24% (Compton 1996 and Etheredge 2003), but there is no evidence that ittranslates in arrhythmia protection. (v) Potassium channel openers:Nicorandil, a potassium channel opener given intravenously at 2-20umol/L appreviates the QT interval in LQT1 and LQT2 subjects.(Shimizu W2000). (vi) hERG current enhancers: RPR 260243 reversesdofetalide-induced action potential prolongation in guinea pig myocytes(Kang J2005). (vii) Calcium channel blockers: Calcium influx throughL-type calcium channels maintains the plateau phase, the duration of theaction potential and the QT interval of the action potential. Verapamilan L-type calcium channel blocker, and inhibitor of I_(Na) abbreviatesthe QT interval and suppresses TdP in LQTS models is used in patientswith paroxysmal atria-ventricular nodal reentrant tachycardia withsignificantly shortened QT at low heart rates. The hERG inhibitory EC₅₀is 83 uM. When verapamil is administered at appropriate dosage, torsadesde points may be avoided (Fauchier L 1999). (viii) Trafficking defectscorrection: Defects in transport of proteins and glycoproteins formingtrans-membrane ion pores in the cardiac cell membrane reduce theamplitude of corresponding currents and have a role in LQTS.Fexofenadine, a metabolite of terfenadine or thapsigargin can rescuesuch defective trafficking without blocking hERG current in selectivemissense mutations associated with LQT2. (ix) Gap Junction couplingenhancers: Gap junctions are intercellular channels allowing both smallmolecules and current to be transferred between cardiac cells. Heartfailure and hypertrophy are associated with uncoupling of gap junctions.Enhancing gap junctions can produce an anti-arrhythmic effect wheredispersion of repolarization is enhanced in LQTS. Infusion of asynthetic peptide, AAP10 a gap junction enhancer reduces the QT intervalin the rabbit left ventricular preparation (Quan XQ, 2007).

This nonclinical laboratory study described in the present invention wasconducted in accordance with the United States Food and DrugAdministration (FDA) Good Laboratory Practice Regulations, 21 CFR Part58, the Organization for Economic Cooperation and Development (OECD)Principals of Good Laboratory Practice [C(97) 186/Final], issuedNovember 26, 1997, and the Japanese Ministry of Health, Labour andWelfare (MHLW) Good Laboratory Practice Standards Ordinance No. 21, Mar.26, 1997.

Study Outline: 1) Test articles: Curcumin, Empty Liposomes, Liposomalcurcumin, (0.014, 0.20, 3.4 and 11.4 μM); 2) Test System:hERG-expressing HEK 293 transfected cell line; 3) Test performed:Whole-cell patch-clamp current acquisition and analysis; 4) ExperimentalTemperature: 35±2° C.

Application of test article: 1) 5 minutes of exposure to eachconcentration in presence of closed circuit perfusion (2 mL/min); 2) 5minutes for washout periods in presence of a flow-through perfusion(2mL/min) in addition to a closed circuit perfusion (2 mL/min); 3) Thepositive controls, (100nM E-4031, and Terfenadine (0.01, 0.03, 0.1 uM)were added to naive cells obtained from the same cell line and samepassage for a period of 5 minutes in presence of a closed circuitperfusion (2 mL/min); 4) Curcumin, Terfenadine and E-4031 were eachvortexed for 15 minutes with empty liposomes, and then tested. Cellswere under continuous stimulation of the pulses protocol throughout thestudies and cell currents were recorded after 5 minutes of exposure toeach condition.

Data acquisition design: Acquisition Rate(s): 1.0 kHz. Design foracquisition when testing the compounds or the vehicle/solventequivalent: 1 recording made in baseline condition, 1 recording made inthe presence of concentration 1, 2, 3 or 4, and 1 recording made afterwashout (only after the fourth concentration). Design for acquisitionwhen testing the positive controls: 1 recording made in baselinecondition, 1 recording made in the presence of the positive control, andn =number of responsive cells patched on which the whole protocol abovecould be applied.

Statistical analysis: Statistical comparisons were made using pairedStudent's t-tests. For the test articles, the currents recorded afterexposure to the different test article concentrations were statisticallycompared to the currents recorded in baseline conditions. Currents,recorded after the washout, were statistically compared to the currentsmeasured after the highest concentration of test article. In the sameway, currents recorded after the positive control, were compared to thecurrents recorded in baseline conditions. Differences were consideredsignificant when p≤0.05.

Data Exclusion criteria: 1) Timeframe of drug exposure not respected; 2)Instability of the seal; 3) No tail current generated by the patchedcell; 4) No significant effect of the positive control; and 5) More than10% variability in capacitance transient amplitude over the duration ofthe study.

The in vitro effects of curcumin, liposomal curcumin, empty liposome andpositive controls E-4031 and Terfenadine were determined on the humandelayed rectifier current using human embryonic kidney (HEK) 293 cellstransfected with the human ether-a-go-go-related gene (hERG).

I_(Kr) channel inhibition and cardiac toxicity has become a majorliability for some classes of drugs, in particular, those that triggerI_(Kr) channel inhibition and/or in vivo QT prolongation. Many drugshave been found to have this activity during preclinical drugdevelopment, which has led to the abandonment of many promising drugclasses. A number of QT-prolonging drugs have been withdrawn or havevery limited use as a result of this activity. Examples include, but arenot limited to: crizotinib, nilotinib, terfenadine, astemizole,gripafloxacin, terodilene, droperidole, lidoflazine, levomethadyl,sertindoyle and cisapride.

There are a great number of drugs that are currently marketed withincreased risk of LQTS and TdP. Some non-limiting examples are presentedbelow:

Aloxi or palonasitron HCL: 5-hydroxytryptamine-3 receptor antagonist, anintravenous drug for post-operative nausea and vomiting.(Eisai Corp.Helsinn, Switz.) AE's include >2% EKG, 5% QT prolongation, 4%bradycardia, at doses above 2.25 mg.

Amiodarone (cordorone X) a Class III antiarrhythmic agent, for WPWsyndrome, for ventricular arrhythmias: Females>males risk regarded aslow. 1-3% have predominantly Class III effects. SA node dysfunction, andenhanced cardiac arrhythmias. MOA is prolongation of myocardialcell—action potential duration, and refractory period, a 10% increase inQT intervals associated with worsening of arrhythmias and TdP, andnoncompetitive α- and β-adrenergic inhibition. QTc prolongation with andwithout TdP with concomitant administration of fluoroquinolones,macrolide antibiotics or azoles. TEVA Pharmaceuticals IND.Ltd.

Arsenic trioxide: an ineffective hERG blocker(IC₅₀>300 uM), may have anindirect effect on hERG current, an anti-cancer drug. The manufactureris Cephalon, Inc.

Astemizole*: a second generation histamine H1 and H3 receptorantagonist, and antmalarial marketed by Janssen. Structurally similar toterfenidine and haloperidol. Originally used for allergic rhinitis: nolonger available in U.S. because of rare but fatal arrhythmias. IC₅₀ is50 nM hERG tail current.

Bepridil: is a low potency long-acting calcium channel blockingagent(EC50 is 10 uM). Both K+ channels are sensitive targets to calciumchannel blockers. It blocks the rapid component hERG in aconcentration-dependent manner (EC50 is 0.55 uM) and also inhibits theKvLQT1/IsK K+ channel which generates the slow components of the cardiacdelayed rectifier K+ current. These changes can lead to long QT. It isalso a calmodulum antagonist with significant anti-effort associatedangina, and antihypertensive activity. Manufacturer TOCRIS BioscienceInc.

Chloroquine: antimalarial: Novartis Pharma AG. Inhibits hERG channels ina concentration and time manner. The half maximal inhibitoryconcentration (IC₅₀) 2.5 uM.

Chlorpheniramine: a low potency first generation antihistamine H1blocker, which induces QT prolongation, i.e., a hERG blocker in aconcentration dependent manner. It affects the channels in the activatedand inactivated states but not in the closed states. Overdose of firstand second generation antihistamines exert arrhythmic effects byaffecting k+ currents.

Chlorpromazine (Thorazine): anti-psychotic/antiemetic/schizophreniadeveloped by Rhone-Poulec in 1950. It causes cardiac arrhythmias (FowlerNO 1976).

Cisapride: used as gastroprokinetic agent by Janssen Inc.: It waswithdrawn in 2000 due to its Long QT side effect (Layton D 2003).

Celaxa (citalopram) a QT prolonger Forest Labs: A selective serotoninreuptake inhibitor (SSRI) which prolongs the QTc interval via directblockade of the potassium hERG channel, disrupts hERG protein expressionin the cell membrane effectively decreasing the number of hERG potassiumchannels and blocks the I-type calcium current leading to prolongeddepolarization. (Witchel, et al).

Clarithromycin and Erythromycin: Antibiotics, females are more sensitivethan males. Both cause QT prolongation and TdP. Erythromycin reducteshERG current in a concentration dependent manner with an IC₅₀ of 38.9,and clarithromycin 45.7 uM at clinically relevant concentrations.

Curcumin (diferuloylmethane): Inhibits hERG current (Moha ou Maati H,2008). Curcumin at IC₅₀ of 3.5 uM is a moderate potency molecule(Katchman A N, 2005).

Disopyramide: A class 1 antiarrythmic drug (Vaughan WilliamsClassification) associated with acquired LQTS. Prolongs the QT intervaland widens the QRS complex QT in a dose dependent fashion (IC₅₀ 7.23uM). Blocks both sodium and potassium channels depresses phase “O”depolarization and prolongs duration of action potential of normalcardiac cells in atrial and ventricular tissues.

Dofetilide: A Class III antiarrhythmic agent marked by Pfizer as Tikosynoral capsules used for maintenance of sinus rhythm and atrialfibrillation. Selectively blocks IKr, the delayed rectifier outwardpotassium current. TdP is a serious side effect with a dose relatedincidence of 0.3-10.5%. This is a twofold increase in death risk ifpre-treatment QTc is greater than 479 ms. A high potency hERG blocker:IC₅₀ is 10 nM.

Domperidone: An antidopaminergic drug used as an antinausea agent. ByJanssen Pharmaceuticals, not available in the U.S. Associated withcardiac arrest and arrhythmias, and increased QT prolongations inneonates (Djeddi D 2008).

Doxorubicin: 30 uM prolongs QTc by 13%; causes acute QT prolongationwithout significantly blocking hERG channels but inhibits IKs (IC₅₀:4.78 uM).

Dronedarone: A non-iodinated analogue of amiodarone.(blocks hERG at IC₅₀of 70 nM), used for over 40,000 patients with atrial fibrillation. Wildtype hERG tails measured at −40 mV following activation at +30 mV wereblocked with IC₅₀ values of 59 nM. hERG inhibition followed channelgating, with block developing on membrane depolarization independent ofchannel activation High external [K+] (94 mM) reduced potency of I(hERG)inhibition and is independent of Y652 and F656 aromatic acid residues.Manufactured by Chemsky (Shanghai) International, and Sanofi-Avantis Incas (Muttag). The UK NIH blocked this drug in 2010 based upon cost.

Droperidol: A central sedative, anti-nausea, anesthesia adjunct,Associated with prolongation of the QT interval, TdP and sudden death.hERG tail currents following test pulses to 50 mV were inhibited with anIC₅₀ of 77.3 nM. hERG channels were affected in their open andinactivated states. Potency was decreased with mutation of Phe-656 tothr or Ser-631 to Ala. Fourteen companies are listed for this compound.

Grepafloxacin: An oral fluoroquinolone antibiotic caused a number ofsevere cardiovascular events including PQTS and was voluntarilywithdrawn from the market. (WHO 1999).

Haldol, Haloperidol: A high potency hERG blocker, antipsychoticschizophrenia, agitation, when given intravenously or at higher thanrecommended doses, risk of sudden death, QT prolongation and TdPincreases. Janssen-Silag Ltd.

Halofantrine: Antimalarial, associated with cardiac arrhythmias andsignificant QT prolongation.females more sensitive than males.Glaxo-Smith-Kline.

Ibutilide: Corvert by Pfizer, a pure class III antiarrythmic for atrialflutter and fibrillation, females more sensitive than males. Inducesslow inward sodium current. Does not block K current, but prolongsaction potential.

Levomethadyl: Opiate agonist/pain control, narcotic dependence. Similarto methadone. Roxanne Labs removed from market because of ventricularrhythm disorders.

Lidoflazine: A piperazine calcium channel blocker with anti-arrhythmicactivity. high potency hERG blocker (IC₅₀ of 16 nM) of the alphasub-unit of the potassium channel. Preferentially inhibits openactivated channels. 13 fold more potent than Verapamil against hERG.

Loratidine, Claritin: A second generation antihistamine, a hERG blockerat an IC₅₀ of 173 nM. may have an indirect effect on hERG repolarizationcurrent. Marked by Schering-Plough.

Lovostatin: A low-potency hERG blocker synthetic.

Mesoridazone: Antipsychotic schizophrenia.

Methadone: Interacts with the voltage—gated myocardial potassiumchannels in a concentration dependent manner causing serious cardiacarrhythmias, and deaths from TdP and ventricular fibrillation inpatients taking methadone. IC₅₀ is 4.8 uM (compared with 427 uM forheroin) an antidopaminergic drug. Methadone related predispositions toTdP are female, high dosages, CYP2 B6 slow metabolizer of S-methadoneand DNA polymorphisms. Parenterol methadone and chlorobutanolcombinations are contraindicated. QT prolonging activity is mainly dueto S-methadone which blocks hERG current 3-5 fold more potently thanR-methadone.

Methanesulphonanilide (E-4031): An extremely high potency compound,inhibits hERG at nM concentrations. Used as positive control in standardassays.

Moxifloxacin: A hERG channel blocker: at 100 uM prolonged QTc by 22% notprevented by dexrazoxane.

Pentamadine: An ineffective hERG blocker (IC₅₀>300 uM), anti-infective,pneumocystis pneumonia. Associated with QT interval lengthening and TdP,hence may have an unknown indirect effect on hERG repolarization.

Pimozide: Antipsychotic, Tourette's tics.

Prenylamine: A moderate hERG blocker.

Probucol: Antilipemic, anticholesterolemic, no longer available in theU.S.

Procainamide: Anti-arrythmic.

Propafenone: A low-potency hERG blocker(IC₅₀>1 uM).

Pyrilamine: A low potency hERG blocker.

Quinidine: Anti-arrythmic females>males.

Seldane (Terfenidine): A high potency hERG blocker.

Sertindole: A moderate potency hERG blocker.

Sotalol: A LQT2 model, action is prevented by nicorandil a potassiumchannel opener. It can act as an antiarrythmic, β-blocker forventricular tachycardia, atrial fibrillation (DucroqJ 2005). Two (2) %of 1288 patients exhibited QT prolongation, and a QTc greater than 455ms lead to TdP.

Sparfloxacin: Antibiotic.

Thioridazine: A moderate potency hERG blocker.

Vandetanib: An oral kinase inhibitor marketed by Astra-Zeneca isapproved for progressive metastatic or locally advanced medullarythyroid cancer. QT prolongation, TdP and sudden death are included in aboxed warning. The most common (>5%) grade ¾ adverse reactions includeQT prolongation fatigue and rash.

Terfenadine an antihistamine prodrug for the active form fexofenadine,and E-4031 were selected as a reference compounds for this study.Terfenadine has reported ventricular arrhythmias cardiotoxic effects,particularly if taken in combination with macrolide antibiotics orketoconazole. An IC₅₀ hERG inhibitory effect value of 99 nM wascalculated from data obtained in the same cell line as that used for thetest article in this study. E-4031, a Class III anti-arrhythmic drug isa synthetic toxin used solely for research purposes with one clinicalexception (Okada Y., 1996). Its mechanism of action is to block the hERGvoltage-gated potassium channels. At 100 nM E-4031 inhibited 90.6% ofthe current density. The inhibitions observed are in line with internalvalidation data generated in identical conditions, and agree withpublished inhibition values for this compound. These results confirm thesensitivity of the test system to hERG-selective inhibitors, in thiscase, Terfenadine and E-4031.

The effect of Curcumin on whole-cell IKr hERG currents: whole-cellcurrents elicited during a voltage pulse were recorded in baselineconditions, following the application of the selected concentrations ofcurcumin and following a washout period. As per protocol, 4concentrations of curcumin were analyzed for hERG current inhibition.The cells were depolarized for one second from the holding potential(−80 mV) to a maximum value of +40 mV, starting at −40 mV andprogressing in 10 mV increments. The membrane potential was thenrepolarized to −55 mV for one second, and finally returned to −80 mV.

Whole-cell tail current amplitude was measured at a holding potential of−55 mV, following activation of the current from −40 to +40 mV. Currentamplitude was measured at the maximum (peak) of this tail current.Current density was obtained by dividing current amplitude by cellcapacitance measured prior to capacitive transient minimization.

Current run-down and solvent effect correction: all data points havebeen corrected for solvent effect and time-dependent current run-down.Current run-down and solvent effects were measured simultaneously byapplying the experimental design in test-article free conditions (DMSO)over the same time frame as was done with the test article. The loss incurrent amplitude measured during these so-called vehicle experiments(representing both solvent effects and time-dependent run-down) wassubtracted from the loss of amplitude measured in the presence of thetest article to isolate the effect of the test article, apart from theeffect of the solvent and the inevitable run-down in current amplitudeover time.

The study presented herein quantified the effect of curcumin solubilizedin DMSO on IKr. The concentrations of curcumin (0.014, 0.2, 3.4 and 11.4μM) were based on information available at the time of the design ofthis study. The concentrations were selected based on: (1) the predictedhuman plasma levels at the planned lowest Phase 1 dose level; (2) thepredicted human plasma concentrations at the planned highest Phase 1dose level; (3) 30-fold over the predicted human therapeutic plasmalevels; and (4) 100-fold over the predicted human therapeutic plasmalevels. These selected concentrations are considered to provide valuablepredictions of the effect of curcumin on human cardiacelectrophysiology. Curcumin 99.2% pure, was synthesized under GMPconditions in Sami Labs, Bangalore, India and stored at 4° C. in theabsence of light. One mL aliquot of each curcumin concentration used toexpose the cells included in this study were independently analyzed forcurcumin content. For the subsequent studies GMP grade liposomalcurcumin was formulated at Polymun GmbH, Vienna Austria, and stored at4° C. The liposomes were obtained from Polymun GmbH, terfenadine andE04031 were purchased from Sigma Aldrich Fine Chemicals.

TABLE 1 Effect of terfenadine, a positive control on hERG currentdensity from transfected HEK 293 cells at 20 mV. Corrected NormalizedNormalized Current Current p Density Density SEM value n = Baseline1.000 1.000 n/a n/a 3 Terfenadine, 0.01 μM* 0.645 0.767 0.090 0.122 3Terfenadine, 0.03 μM** 0.650 0.772 0.073 0.088 3 Terfenadine, 0.1 μM**0.362 0.483* 0.063 0.015 3 *10 nM, **30 nM, ***100 nM. Terfenadineinhibited IKr with an IC₅₀ of 0.065 umolar (65 nM) potency.

TABLE 2 Effect of Terfenadine on hERG current density from transfectedHEK 293 cells at 20 mV. Corrected Normalized Normalized Current Currentp Density Density SEM value n = Baseline 1.000 1.000 n/a n/a 3Terfenadine, 30 nM 0.469 0.548 0.080 0.111 2 Terfenadine, 100 nM 0.3990.478* 0.072 0.018 3 Terfenadine, 300 nM 0.043 0.122* 0.004 0.000 3 *Thecurrent recorded after exposure to the test article concentration wasstatistically different from the current recorded in baseline condition.Difference was considered statistically significant when p ≤ 0.05.

FIG. 1 is a graphical representation of the data presented in Table 2.FIG. 2 is a graph of the current-voltage (I-V) relationship of hERGcurrent amplitude from transfected HEK 293 cells exposed to terfenadine.FIG. 3 is a graph of the effect of terfenadine on hERG current densityfrom transfected HEK 293 cells at 20 mV. FIG. 4 is a graph of the I-Vrelationship of hERG current amplitude from transfected HEK 293 cellsexposed to terfenadine.

TABLE 3 Effect of E-4031 on hERG current density from transfected HEK293 cells at 20 mV. Corrected Normalized Normalized Current Current pDensity Density SEM value n = Baseline 1.000 1.000 n/a n/a 3 E-4031, 100nM 0.124 0.094* 0.067 0.0055 3 E-4031 inhibited IKr with an IC50 of 50nM. FIG. 5 is a graph showing the effect of E-4031 on hERG currentdensity from transfected HEK 293 cells at 20 mV.

TABLE 4 Effect of Curcumin on hERG current density from transfected HEK293 cells at 20 mV. Corrected Normalized Normalized Current Current pDensity Density SEM value n = Baseline 1.000 1.000 n/a n/a 7 Curcumin,0.014 μM 0.892 0.862 0.084 0.1521 7 Curcumin, 0.2 μM 0.773 0.744* 0.0700.0107 7 Curcumin, 3.4 μM 0.642 0.612* 0.095 0.0064 7 Curcumin, 11.4 μM0.234 0.204* 0.016 0.0000 7 Washout 0.489 0.459 0.127 0.2036 3

At a concentration of 11.4 μM curcumin caused 79.6% inhibition of thehERG tail current density at 1+20 (n=7). Paired student's t-testsconfirmed that the difference in normalized current density measured atbaseline and in the presence of 0.2 to 11.4 μM of curcumin reached theselected threshold for statistical significance (p 0.05). Table 3provides p-values obtained from statistical analysis. Fifty percentinhibition of the current was achieved within the range ofconcentrations (0.014 to 11.4 μM) selected for this study. An IC₅₀ valueof 4.9 μM was calculated from the data obtained. FIG. 6 is a graph ofdata shown in Table 4. FIG. 7 is a graph of the I-V relationship of hERGcurrent amplitude from transfected HEK 293 cells exposed to curcumin.

TABLE 5 Effect of Curcumin (as liposomal curcumin) on hERG currentdensity from transfected HEK 293 cells at 20 mV. Corrected NormalizedNormalized Current Current p Density Density SEM value n = Baseline1.000 1.000 n/a n/a 7 Curcumin (liposomal 0.854 0.934 0.039 0.142 7curcumin), (0.014 μM) Curcumin (liposomal 0.838 0.918 0.092 0.408 7curcumin), (0.2 μM) Curcumin (liposomal 0.769 0.848 0.072 0.079 7curcumin), (3.4 μM) Curcumin (liposomal 0.716 0.795* 0.082 0.046 7curcumin), (11.4 μM) Washout 0.474 0.554* 0.101 0.020 4

P-values obtained from statistical analysis indicates borderlinesignificant differences of current density from baseline at 11.4 uM,however the extent of current inhibition was less than the IC₅₀.

FIG. 8 is a graph showing the effect of curcumin (as liposomal curcumin)on hERG current density from transfected HEK 293 cells at 20 mV and FIG.9 is a graph showing the I-V relationship of hERG current amplitude fromtransfected HEK 293 cells exposed to Curcumin (as liposomal curcumin).

In Table 5 the rectifying inward current showed that the inhibitioneffect of curcumin on the hERG tail current is voltage dependent withhigher potency at positive holding potentials. The currents recordedafter washout were compared statistically to the currents recorded afterthe highest concentration of Curcumin (liposomal curcumin) (11.4 μM).

TABLE 6 Effect of empty liposome vortexed with curcumin on hERG currentdensity from transfected HEK 293 cells at 20 mV. Corrected NormalizedNormalized Current Current p Density Density SEM value n = Baseline1.000 1.000 n/a n/a 3 Curcumin (Lipo- 0.937 0.994 0.073 0.946 3 Curc.),0.2 μM Curcumin (Lipo- 0.738 0.796 0.055 0.064 3 Curc.), 3.4 μM Curcumin(Lipo- 0.498 0.555 0.119 0.064 3 Curc.), 11.4 μM Washout 0.479 0.5360.145 0.899 3

Liposome concentration was 0.7,12,41 ng/ml. No significant differencefrom curcumin at any dose level.

FIG. 10 is a graph showing the effect of Curcumin (Liposomes+Curcumin)on hERG current density from transfected HEK 293 cells at 20 mV, andFIG. 11 is a graph of the I-V relationship of hERG current amplitudefrom transfected HEK 293 cells exposed to Curcumin (Liposomes+Curcumin).The current recorded after washout was compared and similarstatistically to the currents recorded after the highest concentrationof curcumin at 11.4 uM. The current IC₅₀ was not reached.

TABLE 7 Effect of Liposomes on hERG current density from transfected HEK293 cells at 20 mV. Corrected Normalized Normalized Current Current pDensity Density SEM value n = Baseline 1.000 1.000 n/a n/a 3 Liposome,0.921 1.041 0.037 0.379 3 0.7227 ng/mL Liposome, 0.805 0.926 0.065 0.3743 12.285 ng/mL Liposome, 0.888 1.009 0.075 0.919 3 41.193 ng/mL Washout0.817 0.938 0.151 0.734 3

Liposomes do not exhibit an inhibitory effect on the in vitro hERGchannel. The current recorded after washout was comparable statisticallyto the currents recorded after the highest concentration of Liposomes(41.193 ng/mL). FIG. 12 is a graph of the data presented in Table 7, andFIG. 13 is a graph of the I-V relationship of hERG current amplitudefrom transfected HEK 293 cells exposed to liposomes.

TABLE 8 Effect of Liposomes + E-4031 on hERG current density fromtransfected HEK 293 cells at 20 mV. Corrected Normalized NormalizedCurrent Current p Density Density SEM value n = Baseline 1.000 1.000 n/an/a 3 Liposome, 0.489 0.610 0.115 0.077 3 0.72 ng/mL + E-4031, 30 nMLiposome, 0.219 0.339* 0.067 0.010 3 12.29 ng/mL + E-4031, 100 nMLiposome, 0.171 0.292* 0.022 0.001 3 41.19 ng/mL + E-4031, 300 nMWashout 0.130 0.251 0.037 0.675 2 *the current recorded after exposureto the test article concentration was statistically different p ≤ 0.05from the current recorded in baseline condition.

FIG. 14 is a graph showing the effect of liposomes+E-4031 on hERGcurrent density from transfected HEK 293 cells at 20 mV. FIG. 15 is agraph of the I-V relationship of hERG current amplitude from transfectedHEK 293 cells exposed to Liposomes+E-4031.

Empty Liposomes when vortexed with E-4031 at 30-300 nM concentrations donot prohibit the anti-hERG effect of E-4031. E-4031 inhibition. Thecurrent recorded after washout was compared statistically to thecurrents recorded after the highest concentration of Liposomes+E-4031.

TABLE 9 Effect of Liposomes + Terfenadine on hERG current density fromtransfected HEK 293 cells at 20 mV. Corrected Normalized NormalizedCurrent Current p Density Density SEM value n = Baseline 1.000 1.000 n/an/a 3 Terfenadine (Liposome + 0.298 0.392* 0.065 0.011 3 Terfenadine),30 nM Terfenadine (Liposome + 0.122 0.216* 0.073 0.008 3 Terfenadine),100 nM Terfenadine (Liposome + 0.117 0.211* 0.032 0.000 4 Terfenadine),300 nM Washout 0.276 0.369 0.017 0.081 2 *Mean that the current recordedafter exposure to the test article concentration was statisticallydifferent from the current recorded in baseline condition. Differencewas considered statistically significant when p ≤ 0.05.

The data presented in Table 9 hereinabove is represented graphically inFIG. 16, and FIG. 17 is a graph showing the I-V relationship of hERGcurrent amplitude from transfected HEK 293 cells exposed toliposomes+terfenadine. There was no effect of empty liposomes whenvortexed with Terfenadine at 30-300 nM the Terfenadine inhibition ofhERG current density.

The data presented hereinabove suggest that curcumin, within the rangeof concentrations tested and in the specific context of this studydown-modulates the IKr current, i.e., it interacts with the proteinsencoded by the hERG gene and activates channel gating functionsdecreasing ion flow. A similar observation with a curcuminoid mixture(78% curcumin) was published (Moha ou Matti, 2008). These data supporttheir initial observation, and emphasize that the curcumin(diferuloylmethane) molecule exhibits the predominant if not all the IKrinhibition.

The findings of the present invention that liposomal curcumin orvortexed mixtures of liposomes with curcumin prohibited IKr downmodulation by curcumin allowing normal gating functions to occur suggestthat liposome encapsulation of curcumin is not necessary to preventinteractions with channel drug receptor sites. The empty liposome didnot appear to interact with the protein encoded by the hERG gene in theabsence of curcumin, or in the presence of E-4031 and terfenadinerelates to questions regarding the specificity and degree of affinitiesor preferential interactions of the receptors in the K+ channel(Zachariae U 2009).

Ikr/hERG suppression induced by curcumin is mitigated when the curcuminis incorporated within a liposome or simply vortexed with it prior toexposure. Combined intravenous administration of this liposome andintravenous QT prolonging drugs other than curcumin may mitigate delayedQT in vivo.

EXAMPLE 2 Liposomes Ameliorate Drug-Induced Inhibition of the CardiacI_(Kr) Channel

Crizotinib (Xalkori®) and nilotinib (Tasigna®) are tyrosine kinaseinhibitors approved for the treatment of non-small cell lung cancer andchronic myeloid leukemia, respectively. Both have been shown to resultin QT prolongation in humans and animals. Liposomes have been shown toameliorate drug-induced effects on the I_(Kr) (K_(v)11.1) channel, codedby the human ether-a-go-go-related gene (hERG). A study was conducted todetermine if liposomes would also decrease the effect of crizotinib andnilotinib on the I_(Kr) channel. Crizotinib and nilotinib were tested ina standard in vitro I_(Kr) assay using human embryonic kidney (HEK) 293cells stably transfected with the hERG. Dose-responses were determinedand 50% inhibitory concentrations (IC₅₀s) were calculated. When the HEK293 cells were treated with crizotinib and nilotinib that were mixedwith liposomes, there was a significant decrease in the I_(Kr) channelinhibitory effects of these two drugs. The use of liposomal encapsulatedQT-prolongation agents, or just mixing these drugs with liposomes, maydecrease their cardiac liability.

Crizotinib (Xalkori®) is an anaplastic lymphoma kinase (ALK) inhibitorapproved for the treatment of non-small cell lung cancer in patientswith ALK-positive tumors. Nilotinib (Tasigna®) is a BCR-ABL kinaseinhibitor approved for Philadelphia chromosome positive chronic myeloidleukemia. Both drugs inhibit the ion channel responsible for thedelayed-rectifier K⁺ current in the heart (k_(r), or K_(v)11.1), encodedby the human ether-a-go-go-related gene (hERG) Inhibition of the I_(Kr)channel can result in prolongation of the QTc, which can lead tolife-threatening polymorphic ventricular tachycardia, or torsades depointes (1). Crizotinib causes QT prolongation in humans and animals,whereas nilotinib has only been shown to cause QT prolongation inhumans.

I_(Kr) channel inhibition and cardiac toxicity can be a major liabilityfor some classes of drugs. Detection of I_(Kr) channel inhibition and/orin vivo QT prolongation during preclinical drug development can lead tothe abandonment of development of promising drugs classes. A number ofQT-prolonging drugs have been withdrawn during development or afterbeing on the market. Examples include terfenadine, astemizole,gripafloxacin, terodilene, droperidole, lidoflazine, levomethadyl,sertindoyle and cisapride.

During development, crizotinib was shown to inhibit the I_(Kr) channelwith a 50% inhibitory concentrations (IC₅₀) of 1.1 μM (2), indicatingthe potential for prolongation of QT interval. The IC₅₀ values werebelow or similar to the C. seen in humans at clinically-relevant doses.Dogs treated intravenously with crizotinib showed decreased heart rateand contractility, and increased left ventricular end diastolicpressure, and increased PR-, QRS- and QT-intervals (3). Thesepreclinical findings correlate with clinical findings of QTcprolongation, bradycardia and cardiac arrest observed occasionally inclinical trials (3,4). Nilotinib was shown to inhibit the I_(Kr) channelwith an IC₅₀ of 0.13 μM (5). But, in contrast to crizotinib, dogstreated orally up to 600 mg/kg did not show QT prolongation (5). Onedifference between the crizotinib and nilotinib dog studies wascrizotinib was given intravenously and nilotinib was given orally. Aswith crizotinib, clinical trials showed an association of therapeuticdoses of nilotinib with QTc prolongation (6,7).

A study by Doherty et al (8) found multiple effects of crizotinib andnilotinib on human cardiomyocytes in vitro. These effects includedcardiac cell death, increased caspase activation, and increasesuperoxide generation. Cardiac cell morphology was altered along withdisruption of normal beat patterns of individual cardiac cells. Forcrizotinib, the cardiac ion channels I_(Kr) NaV1.5 and CaV wereinhibited with IC₅₀s of 1.7, 3.5 and 3.1 μM, respectively. Fornilotinib, IC₅₀s were 0.7, >3 and >3 μM, respectively.

The present inventors have previously shown that liposomes mitigatecurcumin-induced inhibition of the I_(Kr) channel (9). The present studyshows the result on the effects of crizotinib and nilotinib on theI_(Kr) channel, and determine if the addition of liposomes willameliorate these effects.

Crizotinib, at concentrations of 11 and 56 μM, caused 56 and 89%inhibition, respectively, of the I_(Kr) tail current density at 20 mV(FIG. 18A). Paired student's t-tests confirmed that the difference innormalized current density measured at baseline and in the presence of11 and 56 μM of crizotinib reached the selected threshold forstatistical significance (p≤0.05). The IC₅₀ was 8.9 μM with crizotinibalone (Table 10). When crizotinib was vortexed for 10 minutes at roomtemperature with liposomes at a ratio of 9:1 (e.g., 56 μM [25 μg/mL]crizotinib with 225 μg/mL liposomes), only the highest concentration of56 μM crizotinib reached a statistically significant inhibition (59%).The IC₅₀ was 44 μM (Table 10). The liposomes alone did not have anyeffects on the I_(Kr) tail current density (FIG. 18A).

Nilotinib, at concentrations of 0.1 and 1 μM, caused statisticallysignificant inhibition of the I_(Kr) tail current density at 20 mV; 54and 74%, respectively (FIG. 19A). The IC₅₀ was 0.08 μM with nilotinibalone (Table 10). When nilotinib was vortexed for 10 minutes at roomtemperature with liposomes at a ratio of 9:1 (e.g., 1 μM [0.5 μg/mL]nilotinib with 4.5 μg/mL liposomes), there were no effects of nilotinibon the I_(Kr) channel, even at the highest concentrations of 1 μM. TheIC₅₀ was >1 μM (Table 10). The liposomes alone did not have any effectson the I_(Kr) tail current density (FIG. 19A).

The current-voltage relationships of the rectifying inward currentshowed that the inhibitions observed on the tail current were notvoltage dependent for both crizotinib and nilotinib (FIGS. 18B and 19B,respectively).

FIGS. 18A and 18B show I_(Kr) tail current density averages and voltagedependency, respectively, obtained by measuring the I_(Kr) tail peakamplitude at 20 mV in baseline conditions and in the presence ofcrizotinib, liposomes alone, and crizotinib plus liposomes. Forcrizotinib plus liposomes, the crizotinib was mixed with liposomes at9:1 ratio and vortexed; e.g., 56 μM (25 μg/mL) crizotinib with 225 μg/mLliposomes. FIG. 18A is a graph that shows the current density measuredfrom 3 to 4 cells, averaged, normalized against baseline currentdensity, and corrected for time and solvent effects. The values plottedare the mean+standard error of the mean. Statistical comparisons betweenpost-drug exposure and baseline current density levels were made usingrepeat paired Student's t-tests (*). Differences were consideredsignificant when p≤0.05. FIG. 18B is a graph that shows the voltagedependency of the I_(Kr) tail currents inhibition at the higherconcentration of crizotinib tested (56 μM).

FIGS. 19A and 19B show I_(Kr) tail current density averages and voltagedependency, respectively, obtained by measuring the I_(Kr) tail peakamplitude at 20 mV in baseline conditions and in the presence ofnilotinib, liposomes alone, and nilotinib plus liposomes. For nilotinibplus liposomes, the nilotinib was mixed with liposomes at 9:1 ratio andvortexed; e.g., 1 μM (0.5 μg/mL) nilotinib with 4.5 μg/mL liposomes.FIG. 19A is a graph of current density measured from 3 cells, averaged,normalized against baseline current density, and corrected for time andsolvent effects. The values plotted are the mean±standard error of themean. Statistical comparisons between post-drug exposure and baselinecurrent density levels were made using repeat paired Student's t-tests(*). Differences were considered significant when p≤0.05. FIG. 19B is agraph that shows the voltage dependency of the I_(Kr) tail currentsinhibition at the higher concentration of nilotinib tested (1 μM).

TABLE 10 Concentrations that causes fifty percent inhibition (IC₅₀) ofthe I_(Kr) current density in HEK 293 cells stably transfected with thehERG. I_(Kr) IC₅₀ (liposome Test Drug Treatment concentration)Crizotinib Liposomes alone >225 μg/mL Crizotinib 8.9 μM Crizotinib plusliposomes 44 μM (180 μg/mL) Nilotinib Liposomes alone >4.5 μg/mLNilotinib 0.08 μM Nilotinib plus liposomes >1 μM (>4.5 μg/mL)

The positive control, E-4031, produced a statistically significantdecreases in current density at a concentration of 100 nM. E-4031 wastested twice, with results of 67 and 79% inhibitions observed (data notshown). The results were within the range of internal validation datafor this laboratory.

Reagents. Crizotinib and nilotinib were obtained from Reagents Direct.The positive control E-4031(N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbon-yl]phenyl]methanesulfonamide dihydrochloride anhydrous) was obtained from Sigma-Aldrich.The empty liposomes were obtained from Polymun GmbH. The liposomes weremade up of a 9.7:1 ratio of DMPC(1,2-dimyristoil-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]). For crizotinibor nilotinib plus liposomes, the crizotinib or nilotinib was mixed withliposomes at a 9:1 ratio and vortexed for 10 minutes at roomtemperature; e.g., 56 μM (25 μg/mL) crizotinib vortexed with 225 μg/mLliposomes. The internal pipette solution was composed of 140-mM KCl,1.0-mM MgCl₂, 4.0-mM Mg-ATP, 5.0-mM EGTA, 10-mM HEPES, and 10-mMsucrose, pH 7.4±0.05. The hERG external solution was composed of140.0-mM NaCl, 5.0-mM KCl, 1.8-mM CaCl₂, 1.0-mM MgCl2, 10.0-mM HEPES,and 10.0-mM dextrose, pH 7.3±0.05.

Cell Culture. HEK 293 cells stably transfected with the hERG weremaintained in minimum essential medium complemented with 10% fetalbovine serum (Wisent Inc, St. Bruno, Quebec, Canada), 1% minimumessential medium sodium pyruvate, 1% nonessential amino acids, 1%L-glutamine, 1% penicillin/streptomycin, and 400 μg/mL G-418 (Geneticin)as the selection agent (all ingredients from Gibco/Invitrogen,Burlington, Ontario, Canada), and used between passages 12 and 16. Thosecells from which a gigaohm (GD) seal could not be obtained or that didnot generate currents with a distinctive tail current were eliminatedduring the equilibration period.

Procedure. The whole-cell patch-clamp technique was used to functionallyevaluate drug interactions with the ionic channels. HEK 293 cells platedonto 35-mm petri dishes were washed twice with 1 mL of hERG externalsolution followed by the addition of 2 mL of hERG external solution. Thepetri dish was mounted on the stage of an inverted phase contrastmicroscope and maintained at constant temperature (35° C.±2° C.). Aborosilicate glass micropipette filled with the internal pipettesolution was positioned above a single cell using an Eppendorf PatchManmicromanipulator (Eppendorf Canada, Mississauga, Ontario, Canada). Themicropipette was lowered to the cell until a close contact was achieved.The GD-range membrane-pipette seal was then created by applying a slightnegative pressure (resistances were measured using a 5-mV square pulse).Cell capacitance was immediately measured to evaluate cell surface area,using a conversion factor of 1 pF/μm2. This cell surface area was laterused to calculate net current density.

All currents were recorded following analog filtering using a 4-poleBessel filter (Frequency Devices, Haverhill, Massachusetts) set at 1kHz. Through the computer-controlled amplifier, the cell was depolarizedto a maximum value of +40 mV (cultured cells), starting at −10 mV, in 10mV increments, for 1 second. The membrane potential (mV) was thenreturned to −55 mV for 1 second, and finally repolarized to the restingpotential value. This allowed the channels to go from activated toinactivated mode, and back to activated mode, to measure robust tailcurrents. All K+ selective currents passing through hERG channels wererecorded using Axopatch-1D or Axopatch 200B amplifiers and digitizedwith Digidata 1322A or 1440A AD-DA interfaces (Axon instruments Inc,Foster City, Calif., now Molecular Devices Inc). The recording of thecell current started 500 ms before cell depolarization to −40 mV andlasted for 500 ms after the cell had been repolarized to −80 mV.

After baseline recordings were obtained, the increasing concentrationsof the test agents crizotinib and nilotinib, alone or mixed withliposomes, were added in 20-4 aliquots directly to the experimentalchamber and were allowed to disperse through a closed-circuit perfusionsystem using a mini-peristaltic pump (MP-1, Harvard Instruments,Holliston, Massachusetts). Exposure times for each concentration werelimited to 5 minutes. Following the recording of currents in thepresence of the highest concentrations of test agents a flow-throughperfusion system was used to wash out the test article and obtainpostexposure hERG currents in the same manner as previously described.Finally, three naïve cells were exposed to 100 nM of E-4031. Theconcentrations of E-4031 were added into the experimental chamber as wasdone with the test article.

The hERG currents generated by heterologous expression systems such asHEK 293 cells are known to run down over long periods of recording.Therefore, parallel experiments were run in the absence of the testagents and in the presence of the solvent to correct for thetime-dependent decrease in current density, known as current rundown.

Statistical Analysis. The correction for the time-dependent decrease incurrent density involved averaging the changes in current densityassociated with time and solvents, and multiplying the “test-article”results with the resulting correction factor. All results were correctedfor the effect of the vehicle and for time-dependent changes in currentdensity.

hERG current amplitudes are expressed as current density (inanoamperes/picofarad [nA/pF]) to correct for variations in cell sizewithin the population of cells used for this study. Currents wereanalyzed using the Clampfit 10.0 module of the pClamp 10.0 software(Axon Instruments Inc). The results obtained in the presence of eachconcentration were expressed as net current density, normalized againstcurrent density measured in baseline conditions.

The amplitude of the IKr tail current was calculated as the differencebetween the average current recorded before the depolarizing pulse to−40 mV and the maximum transient current recorded at the beginning ofthe repolarizing pulse to −55 mV. Paired t-tests were performed todetermine the statistical significance of the differences in currentdensity obtained before and after the exposure of the cells to the testarticle. Significance was set at P<0.05, where P is the probability thatthe difference in current density levels is due to chance alone.

EXAMPLE 3 In Vivo Evaluation of the Effects of Crizotinib and LiposomesPlus Crizotinib on Cardiac Electrophysiological Parameters of RabbitHearts

In vivo evaluation of the effects of Crizotinib and Liposomes plusCrizotinib on cardiac electrophysiological parameters of rabbit hearts.The purpose of this study is to quantify the in vivo effects ofCrizotinib and liposomes plus Crizotinib on cardiac electrophysiological(PR, QRS, RR, QT, and QTc intervals) parameters from rabbit hearts.

Adult male rabbits weighing between 3 kg and 4 kg. The Crizotinib wastested at 1, 2 and 3 mg/kg (loading doses) over a 10 minute infusionperiod at 0.057 mL/kg/min followed by a 15 minute maintenance infusionof 0.4, 0.8 and 1.2 mg/kg (maintenance doses) respectively at 0.037mL/kg/min. The concentrations were selected based on informationavailable at the time of the design of this study.

The liposomes were injected as an i.v bolus at the ratio of 9:1 of thetotal dose of crizotinib.

TABLE 11 Dose tested Crizotinib Crizotinib Liposomes loading dosemaintenance dose dose (mg/kg) (mg/kg) (mg/kg) 1 0.4 12.6 2 0.8 25.2 31.2 28.8

Anaesthetise rabbits instrumented with ECG leads. The rabbit wasanaesthetized with a mixture of 2.5% isoflurane USP (Abbot Laboratories,Montreal Canada) in 95% O₂ and 5% CO₂. The left jugular vein wascannulated for the i.v infusion of the test article. ECG leads wereplaced on the animal. A continuous recording of the ECG was initiatedand was stopped at the end of the infusion of the last concentration oftest article.

Infusion of each loading dose (1, 2 and 3 mg/kg) lasted 10 minutes andwas followed by a 15 minute infusion of the respective maintenance dose(0.4, 0.8 and 1.2 mg/kg). The infusion rate of the loading dose was0.057 ml/kg/min and 0.037 mg/kg/min for the maintenance dose. Threerabbits were exposed to the crizotinib (n=3).

The liposomes were injected 5 minutes prior to start the infusion ofeach loading dose.

The liposomes were administrated as an i.v bolus in the left ear vein ata ratio of 9:1. Three rabbits were exposed to the liposomes +crizotinib(n=3). Continuous recording of the entire experimental procedure.Significance test performed: Paired Student's t-test with threshold forsignificance set at p≤0.05. n=3.

Crizotinib. Crizotinib caused statistically significant dose dependentdecreases of the heart rate (prolongation of the RR intervals). 2 and 3mg/kg increased the RR intervals by 67 and 110 ms respectively.Crizotinib as of 2 mg/kg caused a statistically significant prolongationof the PR and QRS intervals. The PR interval was increased by 23 ms andthe QRS interval by 13 ms following exposure to 3 mg/kg of crizotinib.Crizotinib caused statistically significant dose dependent prolongationof the QT interval.

When provided at 2 and 3 mg/kg caused a prolongation of the QT intervalsof 34 and 48 ms respectively. Once corrected for change in heart rateusing Van der Water correction factor, Crizotinib still caused astatistically significant prolongation of 38 ms of the QT intervalswhich could not be accounted for by a slower heart rate.

Liposomes plus Crizotinib. The liposomes plus Crizotinib (ratio 9:1) asof 2 mg/kg caused a statistically significant decrease of the heart rate(prolongation of the RR intervals). The RR intervals were increased by61 and 90 ms following exposure to 2 and 3 mg/kg of Crizotinibrespectively.

Liposomes plus Crizotinib caused a prolongation of the PR and QRSintervals. The effect of the liposomes+crizotinib on the PR and QRSintervals was statistically significant at the dose of 3 mg/kg only witha prolongation of 15 ms of the PR interval and 7 ms of the QRS interval.

Liposomes plus Crizotinib at 3 mg/kg caused a prolongation of the QTintervals of 24 ms. However; this effect on the QT intervals was notstatistically significant when compared to the QT interval measured inbaseline conditions. Once corrected for changes in heart rate using Vander Water correction factor, 3 mg/kg caused a prolongation of 15 ms ofQT intervals which was still not statistically significant.

This study aimed at evaluating the effects of Crizotinib and liposomesplus Crizotinib on the cardiac electrophysiological parameters of rabbithearts in vivo. It was found that the Crizotinib alone caused adose-dependent prolongation RR, PR, QRS and QT intervals of the rabbithearts in vivo. The effect of crizotinib on the RR and QT intervals wasstatistically significant as of 1 mg/kg and as of 2 mg/kg on the PR andQRS intervals.

The liposomes plus Crizotinib caused a prolongation RR, PR and QRSintervals of the rabbit hearts in vivo. The effect of liposomes plusCrizotinib on the RR interval was statistically significant as of 2mg/kg while it was statistically significant on the PR and QRS intervalsat 3 mg/kg only. The liposomes plus crizotinib did not cause anystatistically significant prolongation of the QT intervals of the rabbithearts in vivo.

The following table summarizes the index of protection afforded by thepresence of liposomes.

TABLE 12 Comparison of the maximal in vivo effect on theelectrophysiological parameters of the hearts caused by 3 mg/kg ofCrizotinib vs 3 mg/kg of Crizotinib precede by i.v. injection of theliposomes. Liposomes + Crizotinib Crizotinib Protection factor RRinterval 110 91 1.2 prolongation (ms) PR interval 23 15 1.5 prolongation(ms) QRS interval 13 7 1.9 prolongation (ms) QTc interval 38 16 2.4prolongation (ms)

TABLE 13 In vivo effect of Crizotinib on RR interval (ms) of rabbitheart. RR Intervals Condition (ms) SEM p values n = Baseline 257  22.79n/a 3 Crizotinib, 1 mg/kg 294* 27.28 0.040 3 Crizotinib, 2 mg/kg 324*26.12 0.004 3 Crizotinib, 3 mg/kg 367* 34.77 0.013 3 *Mean that thevalue obtained after exposure to the test article concentration wasstatistically different from the value in baseline condition. Differencewas considered statistically significant when p ≤ 0.05.

TABLE 14 In vivo effect of Liposomes plus Crizotinib on RR interval (ms)of rabbit heart. RR Intervals Condition (ms) SEM p values n = Baseline(Liposome) 226  12.48 n/a 3 Liposome + Crizotinib, 1 mg/kg 247  12.120.083 3 Liposome + Crizotinib, 2 mg/kg 287* 13.53 0.003 3 Liposome +Crizotinib, 3 mg/kg 317* 13.97 0.000 3

FIG. 20: In vivo effect of Crizotinib and Liposomes+Crizotinib on RRinterval (ms) of rabbit heart.

TABLE 15 In vivo effect of Crizotinib on PR interval (ms) of rabbitheart. PR Intervals Condition (ms) SEM p values n = Baseline 78 3.71 n/a3 Crizotinib, 1 mg/kg 81 4.14 0.280 3 Crizotinib, 2 mg/kg  92* 4.350.004 3 Crizotinib, 3 mg/kg 101* 8.49 0.048 3

TABLE 16 In vivo effect of Liposomes plus Crizotinib on PR interval (ms)of rabbit heart. PR Intervals Condition (ms) SEM p values n = Baseline(Liposome) 65 2.31 n/a 3 Liposome + Crizotinib, 1 mg/kg 70 3.67 0.096 3Liposome + Crizotinib, 2 mg/kg 75 2.82 0.140 3 Liposome + Crizotinib, 3mg/kg  80* 3.01 0.011 3

FIG. 21: In vivo effect of Crizotinib and Liposomes plus Crizotinib onPR interval (ms) of rabbit heart.

TABLE 17 In vivo effect of Crizotinib on QRS intervals (ms) of rabbitheart. QRS Intervals Condition (ms) SEM p values n = Baseline 43  4.56n/a 3 Crizotinib, 1 mg/kg 48  5.68 0.178 3 Crizotinib, 2 mg/kg 53* 3.440.033 3 Crizotinib, 3 mg/kg 56* 3.85 0.006 3

TABLE 18 In vivo effect of Liposomes plus Crizotinib on QRS intervals(ms) of rabbit heart. QRS Intervals Condition (ms) SEM p values n =Baseline (Liposome) 44 2.03 n/a 3 Liposome + Crizotinib, 1 mg/kg 43 2.360.438 3 Liposome + Crizotinib, 2 mg/kg 45 3.07 0.578 3 Liposome +Crizotinib, 3 mg/kg  51* 2.91 0.039 3

FIG. 22: In vivo effect of Crizotinib and Liposomes plus Crizotinib onQRS interval (ms) of rabbit heart.

TABLE 19 In vivo effect of Crizotinib on QT interval (ms) of rabbitheart. QT Intervals Condition (ms) SEM p values n = Baseline 171  13.75n/a 3 Crizotinib, 1 mg/kg 189* 12.88 0.012 3 Crizotinib, 2 mg/kg 205*12.87 0.016 3 Crizotinib, 3 mg/kg 218* 13.06 0.000 3

TABLE 20 In vivo effect of Liposomes plus Crizotinib on QT interval (ms)of rabbit heart. QT Intervals Condition (ms) SEM p values n = Baseline(Liposome) 175 8.92 n/a 3 Liposome + Crizotinib, 1 mg/kg 181 8.57 0.4153 Liposome + Crizotinib, 2 mg/kg 190 5.10 0.175 3 Liposome + Crizotinib,3 mg/kg 199 4.30 0.074 3

FIG. 23: In vivo effect of Crizotinib and Liposomes plus Crizotinib onQT interval (ms) of rabbit heart.

TABLE 21 In vivo effect of Crizotinib on QTc Van der Water interval ofrabbit heart. QTc interval Condition (ms) SEM p values n = Baseline 235 11.77 n/a 3 Crizotinib, 1 mg/kg 251* 10.55 0.013 3 Crizotinib, 2 mg/kg264* 10.93 0.026 3 Crizotinib, 3 mg/kg 273* 10.18 0.003 3

TABLE 22 In vivo effect of Liposomes + Crizotinib on QTc Van der Waterinterval of rabbit heart. QTc interval Condition (ms) SEM p values n =Baseline 243 7.84 n/a 3 Liposome + Crizotinib, 1 mg/kg 246 7.53 0.526 3Liposome + Crizotinib, 2 mg/kg 252 4.30 0.309 3 Liposome + Crizotinib, 3mg/kg 259 3.58 0.150 3

FIG. 24: In vivo effect of Crizotinib and Liposomes plus Crizotinib onQTc Van der Water intervals of rabbit heart.

EXAMPLE 4 In Vivo Evaluation of the In Vivo Effects of Nilotinib andLiposomes Plus Nilotinib on Cardiac Electrophysiological Parameters ofRabbit Hearts

In vivo evaluation of the In vivo effects of Nilotinib andLiposomes+Nilotinib on cardiac electrophysiological parameters of rabbithearts. The purpose of this study is to quantify the in vivo effects ofNilotinib and liposomes+Nilotinib on cardiac electrophysiological (PR,QRS, RR, QT, and QTc intervals) parameters from rabbit hearts.

Test System. Adult male rabbits weighing between 3 kg and 4 kg.

Doses tested. The Nilotinib was tested at 2, 4 and 5.5 mg/kg (loadingdoses) over a 10 minute infusion period at 0.057 mL/kg/min followed by a15 minute maintenance infusion of 0.14, 0.28 and 0.39 mg/kg (maintenancedoses) respectively at 0.037 mL/kg/min. The concentrations were selectedbased on information available at the time of the design of this study.The liposomes were injected as an i.v bolus at the ratio of 9:1 of thetotal dose of Nilotinib.

Test performed. Anaesthetise rabbits instrumented with ECG leads.Procedure. In vivo rabbit. The rabbits were anaesthetized with a mixtureof 2.5% isoflurane USP (Abbot Laboratories, Montreal Canada) in 95% O₂and 5% CO₂. The left jugular vein was cannulated for IV infusion of thetest agent. ECG leads were placed on the animal, and the ECG signalswere filtered at 500 Hz using an Iso-DAM8A (from Word PrecisionInstrument, Sarasota, Fla., USA) and digitized at a sampling rate of 2.0kHz using a Digidata 1322A interface (from Axon Instruments Inc., FosterCity, Calif., USA, [now Molecular Devices Inc.]). Continuous recordingof the ECG was initiated 5 minutes before beginning infusion of thefirst dose of the compound and was terminated at the end of infusion ofthe last dose. Following baseline ECG recording, the infusion of thefirst loading dose of the compound was started. At the end of the firstloading dose, the infusion was switch to the first maintenance dose. Therabbit was exposed to each dose for 25 minutes (10 minutes of loadingdose followed by 15 minutes of maintenance dose). The same procedure wasapplied until the rabbit was exposed to all of the selected doses oftest agent or vehicle equivalent. The liposomes were injected 5 minutesprior to the start of infusion of each loading dose. The liposomes wereadministrated as an IV bolus in the left ear vein at a ratio of 9:1(μg/mL basis). ECG parameters were analyzed and presented in the samemanner as for the in vitro heart experiment.

TABLE 23 Dose tested Nilotinib loading dose Nilotinib maintenance doseLiposomes dose (mg/kg) (mg/kg) (mg/kg) 2 0.14 25.6 4 0.28 51.1 5.5 0.3970.7

Data analysis and acquisition. Continuous recording of the entireexperimental procedure. Statistical analysis. A paired one-way t-testwas performed to determine the statistical significance of thedifferences in baseline values compared to each treatment. An unpairedone-way t-test, assuming unequal variances, was done to comparecrizotinib or nilotinib alone with crizotinib or nilotinib plusliposomes. Significance test performed: Paired Student's t-test withthreshold for significance set at p≤0.05. n=3.

Nilotinib caused dose dependent decreases of the heart rate(prolongation of the RR intervals). 5.5 mg/kg of Nilotinib caused astatistically significant prolongation of the RR intervals of 78 ms.

Liposomes plus Nilotinib at concentration up to 5.5 mg/kg did not causeany statistically significant effect on the PR intervals.

As of 2 mg/kg Nilotinib caused a statistically significant prolongationof the QRS intervals. 5.5 mg/kg of Nilotinib caused a prolongation ofthe QRS intervals of 7 ms.

Nilotinib caused statistically significant dose dependent prolongationof the QT interval. 4 and 5.5 mg/kg caused a prolongation of the QTintervals of 41 and 66 ms respectively. Once corrected for change inheart rate using Van der Water correction factor, Nilotinib still causeda statistically significant prolongation of the QT intervals which couldnot be accounted for by a slower heart rate.

Liposomes plus Nilotinib. The liposomes plus Nilotinib (ratio 9:1) at5.5 mg/kg caused a statistically significant decrease of the heart rate(prolongation of the RR intervals). The RR intervals were increased by69 ms following exposure to 5.5 mg/kg of Nilotinib.

Liposomes plus Nilotinib at concentration up to 5.5 mg/kg did not causeany statistically significant effect on the PR, QRS and QT intervals.

This study evaluated the effects of Nilotinib and Liposomes plusNilotinib on the cardiac electrophysiological parameters of rabbithearts in vivo.

It was found that the Nilotinib alone caused a dose-dependentprolongation RR, QRS and QT intervals of the rabbit hearts in vivo. Theeffect of Nilotinib on the RR intervals was statistically significant at5.5 mg/kg only while it was statistically significant the QRS and QTintervals as of 2 mg/kg.

The Liposomes plus Nilotinib caused a prolongation RR interval of therabbit hearts in vivo. The effect of liposomes plus Nilotinib on the RRinterval was statistically significant at 5.5 mg/k. The liposomes plusNilotinib did not cause any statistically significant prolongation ofthe PR, QRS and QT intervals of the rabbit hearts in vivo.

The following table summarizes the index of protection afforded by thepresence of liposomes.

TABLE 24 Comparison of the maximal in vivo effect on theelectrophysiological parameters of the hearts caused by 4 mg/kg ofNilotinib vs 4 mg/kg of Nilotinib preceded by i.v. injection of theliposomes. Liposomes plus Protection Nilotinib Nilotinib factor RRinterval prolongation (ms) 54 37 1.5 QRS interval prolongation (ms) 6 32 QTc interval prolongation (ms) 36 8 4.5

TABLE 25 In vivo effect of Nilotinib on RR interval (ms) of rabbitheart. RR Intervals Condition (ms) SEM p values n = Baseline 253 15.10n/a 3 Nilotinib, 2 mg/kg 273 4.68 0.265 3 Nilotinib, 4 mg/kg 307 9.310.132 3 Nilotinib, 5.5 mg/kg  331* 2.70 0.047 3 *Mean that the valueobtained after exposure to the test article concentration wasstatistically different from the value in baseline condition. Differencewas considered statistically significant when p ≤ 0.05.

TABLE 26 In vivo effect of Liposomes plus Nilotinib on RR interval (ms)of rabbit heart. RR Intervals Condition (ms) SEM p values n = Baseline(Liposome) 213 12.00 n/a 3 Liposome plus Nilotinib, 2 mg/kg 217 5.890.590 3 Liposome plus Nilotinib, 4 mg/kg 250 14.66 0.068 3 Liposome plusNilotinib, 5.5 mg/kg  282* 21.52 0.041 3

FIG. 25 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on RR interval (ms) of rabbit heart.

TABLE 27 In vivo effect of Nilotinib on PR interval (ms) of rabbitheart. PR Intervals Condition (ms) SEM p values n = Baseline 79 7.85 n/a3 Nilotinib, 2 mg/kg 78 5.74 0.632 3 Nilotinib, 4 mg/kg 80 5.00 0.947 3Nilotinib, 5.5 mg/kg 82 6.84 0.159 3

TABLE 28 In vivo effect of Liposomes plus Nilotinib on PR interval (ms)of rabbit heart. PR Intervals Condition (ms) SEM p values n = Baseline(Liposome) 65 2.22 n/a 3 Liposome plus Nilotinib, 2 mg/kg 65 0.91 0.8903 Liposome plus Nilotinib, 4 mg/kg 68 1.15 0.076 3 Liposome plusNilotinib, 5.5 mg/kg 71 1.42 0.055 3

FIG. 26 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on PR interval (ms) of rabbit heart.

TABLE 29 In vivo effect of Nilotinib on QRS intervals (ms) of rabbitheart. QRS Intervals Condition (ms) SEM p values n = Baseline 40  5.96n/a 3 Nilotinib, 2 mg/kg 44* 6.66 0.046 3 Nilotinib, 4 mg/kg 46* 6.230.014 3 Nilotinib, 5.5 mg/kg 47* 6.07 0.000 3

TABLE 30 In vivo effect of Liposomes plus Nilotinib on QRS intervals(ms) of rabbit heart. QRS Intervals Condition (ms) SEM p values n =Baseline (Liposome) 36 1.35 n/a 3 Liposome plus Nilotinib, 2 mg/kg 371.74 0.069 3 Liposome plus Nilotinib, 4 mg/kg 39 2.14 0.086 3 Liposomeplus Nilotinib, 5.5 mg/kg 40 2.01 0.069 3

FIG. 27 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on QRS interval (ms) of rabbit heart.

TABLE 31 In vivo effect of Nilotinib on QT interval (ms) of rabbitheart. QT Intervals Condition (ms) SEM p values n = Baseline 161  2.26n/a 3 Nilotinib, 2 mg/kg 182* 4.14 0.042 3 Nilotinib, 4 mg/kg 201* 5.170.025 3 Nilotinib, 5.5 mg/kg 227* 12.37 0.040 3

TABLE 32 In vivo effect of Liposomes plus Nilotinib on QT interval (ms)of rabbit heart. QT Intervals Condition (ms) SEM p values n = Baseline(Liposome) 153 6.73 n/a 3 Liposome plus Nilotinib, 2 mg/kg 148 1.530.473 3 Liposome plus Nilotinib, 4 mg/kg 164 9.17 0.429 3 Liposome plusNilotinib, 5.5 mg/kg 185 9.83 0.089 3

FIG. 28 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on QT interval (ms) of rabbit heart.

TABLE 33 In vivo effect of Nilotinib on QTc Van der Water interval ofrabbit heart. QTc Intervals Condition (ms) SEM p values n = Baseline226  1.27 n/a 3 Nilotinib, 2 mg/kg 245* 3.91 0.029 3 Nilotinib, 4 mg/kg262* 4.42 0.016 3 Nilotinib, 5.5 mg/kg 285* 12.14 0.039 3

TABLE 34 In vivo effect of Liposomes plus Nilotinib on QTc Van der Waterinterval of rabbit heart. QTc Intervals Condition (ms) SEM p values n =Baseline (Liposome) 221 5.73 n/a 3 Liposome plus Nilotinib, 2 mg/kg 2161.25 0.409 3 Liposome plus Nilotinib, 4 mg/kg 230 8.71 0.531 3 Liposomeplus Nilotinib, 5.5 mg/kg 247 8.69 0.113 3

FIG. 29 is a graph that shows the in vivo effect of Nilotinib andLiposomes plus Nilotinib on QTc Van der Water intervals of rabbit heart.

Tables 35 and 36 summarize the results above, and provide furtherinformation about the effects of the present invention.

TABLE 35 Concentrations that caused fifty percent inhibition of theI_(Kr) current density in HEK 293 cells stably transfected with thehERG. Drug Treatment Crizotinib Nilotinib Liposomes alone >225μg/mL^(a) >4.5 μg/mL^(a) Drug alone 8.9 μM 0.08 μM Drug plus Liposomes44 μM >1 μM The concentrations that caused fifty percent inhibition ofthe I_(Kr) current density (IC₅₀) were calculated from the datapresented in FIGS. 18A-B and 19A-B. ^(a)225 and 4.5 μg/mL were thehighest concentrations of liposomes alone tested in the assay, forcrizotinib and nilotinib, respectively.

TABLE 36 Effects of crizotinib and nilotinib, alone and with liposomes,on left ventricular pressure, in in vitro rabbit hearts. Concentrationof Drug Liposomes Liposomes (μM) alone Drug alone plus Drug Crizotinib11 1.25 (2.10) −7.34 (6.19) −1.03 (0.62) 56 0.67 (1.87) −8.22 (6.23)−1.15 (0.37) Nilotinib 14 −0.98 (1.56)  −0.45 (1.51) −0.32 (0.93) 280.47 (2.01) −9.80 (0.19) −3.30 (0.34)

Values are the change from baseline (mmHg).

Values are the mean (SEM), of 3 hearts per group.

In Vitro I_(Kr) Current.

It was found that Crizotinib, at concentrations of 11 and 56 μM, caused57 and 89% inhibition, respectively, of the I_(Kr) tail current densityat 20 mV (FIG. 18A). Paired student's t-tests showed that the differencein normalized current density measured at baseline and in the presenceof 11 and 56 μM of crizotinib reached the selected threshold forstatistical significance (p 0.05). The IC₅₀ was 8.9 μM with crizotinibalone (Table 35). When crizotinib was mixed with liposomes at a ratio of9:1, only the highest concentration of 56 μM crizotinib reached astatistically significant inhibition compared to baseline (59%). TheIC₅₀ was 44 μM. Liposomes plus crizotinib at 11 μM did not have anyeffects on the I_(Kr) tail current density. Liposomes plus crizotinib at56 μM did have a significant effect on the I_(Kr) tail current densitywhen compared to baseline. However, when comparing the current densitybetween crizotinib at 11 and 56 μM, and liposomes plus crizotinib, therewas a significant inhibition of the effects of crizotinib when mixedwith liposomes. The liposomes alone did not have any effects on theI_(Kr) tail current density (FIG. 18A).

Nilotinib, at concentrations of 0.1 and 1 μM, caused statisticallysignificant inhibition of the I_(Kr) tail current density at 20 mV, whencompared to baseline; 54 and 74%, respectively (FIG. 19A). The IC₅₀ was0.08 μM with nilotinib alone (Table 35). When nilotinib was vortexed for10 minutes at room temperature with liposomes at a ratio of 9:1, therewere no effects of nilotinib on the I_(Kr) channel, even at the highestconcentrations of 1 μM. The IC₅₀ was >1 μM. When comparing the currentdensity between nilotinib, and liposomes plus nilotinib, there was asignificant inhibition of the effects of 0.1 and 1 μM nilotinib whenmixed with liposomes. The liposomes alone did not have any effects onthe I_(Kr) tail current density (FIG. 19A).

The current-voltage relationships of the rectifying inward currentshowed that the inhibitions observed on the tail current were notvoltage dependent for both crizotinib and nilotinib (FIGS. 18B and 19B,respectively).

The positive control, E-4031, produced a statistically significantdecreases in current density at a concentration of 100 nM. E-4031 wastested twice, with results of 67 and 79% inhibitions observed (data notshown).

In Vitro Rabbit Heart QTc Intervals.

Crizotinib, at concentrations of 11 and 56 μM, caused a dose dependentprolongation of the QTc interval (FIG. 30A). Mixing crizotinib withliposomes at a ratio of 9:1, resulted in a significant inhibition of thecrizotinib-induced QTc prolongation. Nilotinib, at concentrations of 14and 28 μM, also caused a dose dependent prolongation of the QTc interval(FIG. 30B). As with crizotinib, mixing nilotinib with liposomes,resulted in a significant inhibition of the nilotinib-induced QTcprolongation. The cisapride positive control showed the expectedprolongation of the QTc interval.

The effects of crizotinib and nilotinib on ECGs were associated witheffects on LVP (Table 36). When hearts were exposed to crizotinib ornilotinib alone, there was a decrease in LVP. When liposomes were mixedwith crizotinib or nilotinib, the effects on LVP were reversed.

Rabbit QTc Intervals after In Vivo Dosing.

Rabbits given crizotinib at 1, 2 and 3 mg/kg by IV infusions over 10minutes, followed by a maintenance dose for 15 minutes, showed adose-dependent prolongation of the QTc interval (FIG. 23). Injectingliposomes 5 minutes prior to treatment with crizotinib resulted in asignificant inhibition of the crizotinib-induced QTc prolongation.Rabbits given nilotinib at 2, 4 and 5.5 lmg/kg by IV infusions over 10minutes, followed by a maintenance dose for 15 minutes, showed adose-dependent prolongation of the QTc interval (FIG. 28). As withcrizotinib, injecting liposomes 5 minutes prior to treatment withnilotinib, resulted in a significant inhibition of the nilotinib-inducedQTc prolongation.

These data demonstrated that liposomes protect against the inhibitoryeffect of these kinase-inhibitor drugs on the I_(Kr) channel usingstably hERG transfected HEK 293 cells, and ameliorate cardiac QTcprolongation resulting from both in vitro and in vivo exposure. Theseresults demonstrate that mixing of these drugs with liposomes preventsinteractions of these inhibitory drugs with the I_(Kr) channel allowingmore normal gating kinetics to occur, and decreasing the degree andincidence of QTc prolongation that may occur in the clinic.

Other tyrosine kinase inhibitors have also been shown to have effects onthe QTc interval, including lapatinib, sunitinib and vandetanib (Shah etal., 2013). The most studied in vitro is lapatinib (Lee et al., 2010).Lapatinib was shown to prolong action potential duration of isolatedrabbit Purkinje fibers at 5 μM. This was associated with an inhibitoryeffect on the I_(Ke) channel with an IC₅₀ of 0.8 μM, and a slight effecton the I_(Ks) amplitude at 5 μM. No effects were observed on the I_(Na),I_(K1) or I_(Ca) channels.

In the clinic, crizotinib is given at doses as high as 500 mg/day (250mg twice a day [BID]), which is about 4.2 mg/kg or 156 mg/m², BID. Fromthe FDA's review of the new drug application for crizotinib, steadystate C_(max) in cancer patients given 500 mg BID averaged 650 ng/mL, or1.5 μM (Xalkori, 2011b). MossÉ et al. (2013) reported steady stateC_(max) in children with cancer to be 630 ng/mL (1.4 μM) after dosing280 mg/m² BID. This is well within the range of effects on the in vitroI_(Kr) channel with an IC₅₀ of 8.9 μM reported in the represent study,and 1.1 μM reported during the development of crizotinib (Xalkori, 2011a). Nilotinib is dosed as high as 600 mg/day (300 mg BID), which isabout 5 mg/kg or 188 mg/m², BID. Cancer patients given 400 mg BID hadsteady state C_(max) of 1754 ng/mL, or 3.3 μM (Kim et al., 2011).Chinese patients given 400 mg BID had steady state C_(max) 2161 ng/mL,or 4.1 μM (Zhou et al., 2009). The present study showed an IC₅₀ in theI_(Kr) assay of 0.08 μM, and 0.13 μM was reported during the developmentof nilotinib (Tasigna®, 2007a).

It has previously been reported that liposomes mitigate inhibitoryeffects of curcumin on the I_(Kr) channel (Helson et al., 2012).Curcumin alone inhibited the I_(Kr) channel with an IC₅₀ of 4.9 μM, withthe highest concentration tested (11.4 μM) resulting in 80% inhibition.When mixed with the same liposomes, and same ratio, as in the presentstudy, the highest dose of curcumin tested (11.4 μM) only reached 45%inhibition. Curcumin that was encapsulated in the liposomes, and notjust mixed, also resulted in inhibition of curcumin-induced I_(Kr)inhibition; 25% inhibition at the high concentration of 11.4 μM. In thisstudy, when the positive control E-4031 was tested alone, the IC₅₀ was56 nM. When E-4031 was mixed with liposomes, the IC₅₀ increased to 210nM.

Tartar emetic is a trivalent antimonial drug that causes QT intervalelongation in rats and humans. When tartar emetic was encapsulated inliposomes, the QT effects were abolished (Maciel et al., 2010). Oneimportant difference between the tartar emetic study and the presentstudy is the composition of the liposomes that were used. The liposomesused in the tartar emetic study were composed ofL-a-distearoylphosphatidylcholine, cholesterol and polyethylene glycol2000 distearoylphosphatidyl-ethyanolamine. Another difference is thepresent study showed that simply mixing the drugs with the liposomes, orinjecting them prior to treatment with QT-prolonging drugs, and notencapsulating them, resulted in the inhibitory effects.

One clinical trial in healthy volunteers has shown that encapsulationwith liposomes abolished QT-prolongation effects. When bupivacaine,which increases QT interval in humans and laboratory animals, isencapsulated in liposomes (Exparel®), it did not cause QT prolongationat doses as high as 750 mg given subcutaneously (Naseem et al., 2012).As with the tartar emetic study, here the drug was encapsulated and thecomponents of the liposome were different than the in the present study:cholesterol, 1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol),tricaprylin, and 1,2-dierucoylphosphatidylcholine.

The in vitro assay assessing the effects of drugs on the I_(Kr) (hERG)current is used extensively to help predict potential effects of a drugon QTc interval in the clinic (Witchel, 2001). This has been a usefulassay, but sometime results in false positives. The present studydemonstrates an example where this in vitro assay was very predictive ofin vivo QTc prolongation in both animals and humans.

Surprisingly, based upon the data in the present study, and the datawith curcumin (Helson et al., 2012), it does not appear necessary toencapsulate a drug in the DMPC/DMPG liposome to mitigate I_(Kr)suppression by crizotinib and nilotinib, and possibly otherQTc-prolonging agents. A simple mixing of the compound with theliposomes may be sufficient. The present invention demonstrates that fororally administered QT-prolonging agents, a concurrent subcutaneousadministration of an extended release formulation of liposomes maysuffice. Using the methods and techniques demonstrated herein it ispossible to study QT-prolonging drugs in in vivo animal models of QTcprolongation.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), propertie(s), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skilled in the art recognizethe modified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least +1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

Additionally, the section headings herein are provided for consistencywith the suggestions under 37 CFR 1.77 or otherwise to provideorganizational cues. These headings shall not limit or characterize theinvention(s) set out in any claims that may issue from this disclosure.Specifically and by way of example, although the headings refer to a“Field of Invention,” such claims should not be limited by the languageunder this heading to describe the so-called technical field. Further, adescription of technology in the “Background of the Invention” sectionis not to be construed as an admission that technology is prior art toany invention(s) in this disclosure. Neither is the “Summary” to beconsidered a characterization of the invention(s) set forth in issuedclaims. Furthermore, any reference in this disclosure to “invention” inthe singular should not be used to argue that there is only a singlepoint of novelty in this disclosure. Multiple inventions may be setforth according to the limitations of the multiple claims issuing fromthis disclosure, and such claims accordingly define the invention(s),and their equivalents, that are protected thereby. In all instances, thescope of such claims shall be considered on their own merits in light ofthis disclosure, but should not be constrained by the headings set forthherein.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES—EXAMPLE 1

U.S. Patent Application Publication No. 2010/0004549: System and Methodof Serial Comparison for Detection of Long QT Syndrome (LQTS).

U.S. Patent Application Publication No. 2008/0255464: System and Methodfor Diagnosing and Treating Long QT Syndrome.

U.S. Patent Application Publication No. 2007/0048284: Cardiac ArrhythmiaTreatment Methods.

U.S. Patent Application Publication No. 2001/00120890: Ion ChannelModulating Activity I.

Anderson CL, Delisle B P, Anson B D, et al.: Most LQT2 Mutations ReduceKv11.1 (hERG) Current by a Class 2 (Trafficking Deficient) Mechanism.Circulation 2006 113:365-373.

Compton S J, Lux R L, Ramsey M R, et al.: Genetically defined therapy ofinherited long QT syndrome. Correction of abnormal repolarization bypotassium. 1996 94:1018-1022.

Djeddi D, Kongolo G, Lefaix C, Mounard J, Leke A: Effect of domperidoneon QT interval in neonates. J Pediatrics 2008 153(5): 596-598.

Ducroq J., Printemps R, Le Grand M.: Additive effects ziprasidone and D,L-sotalol on the action potential in rabbit purkinje fibers and on thehERG potassium current. J.Pharmacol. Toxicol Methods 2005 52:115-122.

Etheridge S P, Compton S J, Tristani-Firouzi M, Mason J W: A new oraltherapy for long QT syndrome: long term oral potassium improvesrepolarization in patients with hERG mutations. JAM Coll Cardiol 200342:1777-1782.

Fauchier L, Babuty D Poret P, Autret M L, Cosnay P, Fauchier J P: Effectof Verapamil on QT interval dynamicity. AM J Cardiol. 1999 83(5):807-808A10-1.

Fowler N O, McCall D, Chou T C, Hilmes J C, Hanenson I B,:Electrocardiographic changes and cardiac arrhythmias in patientsreceiving psychotropic drugs. Am J Cardiol 1976 37(2): 223-230.

Jervell A, Lang-Nielson F: Congenital deaf-mutism, functional heartdisease with prolongation of the QT interval and sudden death. Am HeartJ. 1957 54: 59-68.

Kang J, Chen X L, Wang H, et al.: Discovery of a small moleculeactivator of the human ether-a-go-go-related gene(HERG) cardiac K+channel. Mol Pharmacol 2005 67: 827-836.

Katchman A N, Koerner J, Tosaka T, Woosley R L, Eberty S N: Comparativeevaluation of HERG currents and QWT intervals following challenge withsuspected torsadogenic and non-torsdogenic drugs. J Pharmacol Exp Ther.2006 316(3):1098-1106.

Layton D, Key C, Shakir S A: Prolongation of the QT interval and cardiacarrhythmias associated with cisapride: limitations of thepharmacoepidemiological studies conducted and proposals for the future.Pharmacoepidemiol Drug Saf. 2003 12(1):31-40.

Maciel N R, Reis P G, Kato K C et al: Reduced cardio-vascularalterations of tarter emetic administered in long-circulating liposomesin rats. Toxicology Letters. 2010 199 (3): 234-238.

Mehta R T, Hopfer R L, Gunner L A, Juliano R L, Lopez-Berestein G:Formulation, toxicity ,and antifungal activity in vitro ofliposomal-encapsulated nystatin as therapeutic agent for systemiccandidiasis. Antimicrob Agents Chemother. 1987 31(12):1897-1900.

Moha ou Maati H, Ducroq J, Rivet J Faivre J. F. Le Grande M, Bois P:Curcumin blocks the recombinant human cardiac KCNQ1/KCNE1 channels (IKs)stably expressed in HEK 293 cells. Congress de Physiologie,dePharmacologie et de Therapeutique, Clermont-Ferrand, France, 9-11 Avril.2008 Fund. Clin. Pharmacol. 22(Supp1.1).

Shimizu W Antzelevitch C: Sodium channel block with mexiletine iseffective in reducing dispersion of repolarization and preventingtorsade de pointes in LQT2 and LQT3 models of the long QT syndrome. 1997Circulation 96: 2038-2047.

Shimizu W Antzelevitch C: Effects of a K(+) channel opener to reducetransmural dispersion of repolarization and prevent torsade de pointesLQT1, LQT2, and LQT3 models of the long QT syndrome. Circulation, 2000102: 702-712.

Stansfeld P J, Gedeck P, Gosling M, Cox B, Mitcheson J S, Sutclif M J:Drug block of the hERG potassium Channel: insight from modeling.Proteins 2007 68(2): 568-580.

Quan X Q, Bai R, Liu N, Chen B D, Zhang C T. Increasing gap junctioncoupling reduces transmural dispersion of repolarization and preventstorsades de points in rabbit LQT3 model. J Cardiovasc Electrophysiol2007 18:1184-1189.

Zavhariae U, Giordanetto F, Leach A G: Side chain flexabilities in thehuman ether-a-go-go related potassium channel (hERG) together withmatched-pair binding studies suggest a new binding mode for channelblockers. J Med Chem 2009 52(14): 4266-4276.

Zhou Z, Gong Q, January C T: Correction of defective protein traffickingof a mutant HERG potassium channel in human long QT syndrome:Pharmacological and temperature effects. J Biol Chem.1999 274:31123-31126.

REFERENCES—EXAMPLE 2

1. Yap Y G, Camm A J. Drug induced QT prolongation and torsades depointes. Heart. 2003; 89:1363-1372.

2. FDA Pharmacology Review of Xalkori® (crizotinib), IND No. 202570,2011a,www.accessdata.fda.gov/drugsatfda_docs/nda/2011/202570Orig1s000PharmR.pdf(accessed Oct. 9, 2013).

3. FDA Clinical Pharmacology and Biopharmaceutics Review of Xalkori®(crizotinib), IND No. 202570, 2011b,www.accessdata.fda.gov/drugsatfda_docs/nda/2011/202570Orig1s000ClinPharmR.pdf(accessed Oct. 9, 2013).

4. Xalkori (2013) [package insert], Pfizer Laboratories, New York, N.Y.,revised February 2013.

5. FDA Pharmacology Review of Tasigna® (nilotinib), IND No. 22-068,2007a,www.accessdata.fda.gov/drugsatfda_docs/nda/2007/022068s000_PharmR_P1.pdfandwww.accessdata.fda.gov/drugsatfda_docs/nda/2007/022068s000_MedR_P2.pdf,(accessed Oct. 25, 2013).

6. FDA Clinical Pharmacology and Biopharmaceutics Review of Tasigna®(nilotinib), IND No. 22-068, 2007b,www.accessdata.fda.gov/drugsatfda_docs/nda/2007/022068s000_ClinPharmR.pdf,(accessed Oct. 24, 2013).

7. Tasigna (2013), Package insert, Novartis Pharmaceuticals, EastHanover, N.J., revised September 2013.

8. Doherty K R, Wappel R L, Talbert D R, Trusk P B, Moran D M, Kramer JW, Brown A M, Shell S A, Bacus S. Multi-parameter in vitro toxicitytesting of crizotinib, sunitinib, erlotinib, and nilotinib in humancardiomyocytes. Toxicol Appl Pharmacol. 2013; 272(1):245-55.

9. Helson L, Shopp G, Bouchard A, Majeed M. Liposome mitigation ofcurcumin inhibition of cardiac potassium delayed-rectifier current. JRecep Lig Channel Res. 2012; 5:1-8.

10. Maciel N R, Reis P G, Kato K C, et al. Reduced cardio-vascularalterations of tarter emetic administered in long-circulating liposomesin rats. Toxicol Lett. 2010; 199(3): 234-238.

11. Naseem A, Harada T, Wang D, Arezina R, Lorch U, Onel E, Camm A J,Taubel J. Bupivacaine extended release liposome injection does notprolong QTc interval in a thorough QT/QTc study in healthy volunteers. JClin Pharmacol. 2012; 52(9):1441-7.

ADDITIONAL REFERENCES

Crouch, M. A., Limon, L., and Cassano, A. T. (2003). Clinical relevanceand management of drugs-related QT interval prolongation.Pharmacotherapy. 23(7):881-908.

Kim, K. P., Ryu, M. H., Yoo, C., et al. (2011). Nilotinib in patientswith GIST who failed imatinib and sunitnib: importance of prior surgeryon drug availability. Cancer Chemother. Pharmacol. 68(2):285-291.

Lee, H. A., Kim, E. J., Hyun, S. A., Park, S. G., and Kim, K. S. (2010).Electrophysiological effects of the anti-cancer drug lapatinib oncardiac repolarization. Basic Clin. Pharmacol. Toxicol. 107(1):614-618.

Mossé, Y. P., Lim, M. S., Voss, S. D., et al. (2013). Safety andactivity of crizotinib for pediatric patients with refractory solidtumors or anaplastic large-cell lymphoma: a Children's Oncology Groupphase 1 consortium study. Lancet Oncol. 14(16): 472-480.

Shah, R. R., Morganroth, J., and Shah, D. R. (2013). Cardiovascularsafety of tyrosine kinase inhibitors: with a special focus on cardiacrepolarization (QT interval). Drug Saf. 36(5)295-316.

Tasigna®. (2007a). FDA Pharmacology Review of Tasigna® (nilotinib), INDNo. 22-068,http://www.accessdata.fda.gov/drugsatfda_docs/nda/2007/022068s000_PharmR_P1.pdfandhttp://www.accessdata.fda.gov/drugsatfda_docs/nda/2007/022068s000_MedR_P2.pdf,(accessed Oct. 25, 2013).

Tasigna. (2007b). FDA Clinical Pharmacology and Biopharmaceutics Reviewof Tasigna (nilotinib), IND No. 22-068,http://www.accessdata.fda.gov/drugsatfda_docs/nda/2007/022068s000_ClinPharmR.pdf,(accessed Oct. 24, 2013).

Van de Water, A., Verheyen, J., Xhonneux, R., and Reneman, R. S. (1989).An improved method to correct the QT interval of the electrocardiogramfor changes in heart rate. J. Pharmacol. Methods, 22, 207-217.

Witchel, H. J. (2011). Drug-induced hERG block and QT syndrome.Cardiovasc Ther. 29(4):251-259.

Xalkori®, (2011a). FDA Pharmacology Review of Xalkori® (crizotinib), INDNo. 202570,www.accessdata.fda.gov/drugsatfda_docs/nda/2011/202570Orig1s000PharmR.pdf(accessed Oct. 9, 2013).

Xalkori. (2011b). FDA Clinical Pharmacology and Biopharmaceutics Reviewof Xalkori (crizotinib), IND No. 202570,www.accessdata.fda.gov/drugsatfda_docs/nda/2011/202570Orig1s000ClinPharmR.pdf(accessed Oct. 9, 2013).

Zhou, L., Meng, F., Yin, O., et al. (2009). Nilotinib forimatinib-resistant or -intolerant chronic myeloid leukemia in chronicphase, accelerated phase, or blast crisis: a single- and multiple-dose,open-label pharmacokinetic study in Chinese patients. Clin. Ther.31(7):1568-1575.

What is claimed is:
 1. A composition for treating or preventing one ormore cardiac channelopathies or conditions resulting from irregularitiesor alterations in cardiac patterns, or both, in a human or animalsubject consisting of: one or more pharmacologically active agentsprovided in an amount that inhibits the activity of anether-a-go-go-related gene (hERG), wherein the one or morepharmacologically active agents are kinase inhibitors selected fromcrizotinib and nilotinib, wherein the amount of the one or morepharmacologically active agents is sufficient to treat a cancer, whereinthe cancer is selected from non-small cell lung cancer or chronicmyeloid leukemia; one or more empty liposomes, wherein the emptyliposomes are administered prior to, concomitantly, or afteradministration of the pharmacologically active agent, wherein the one ormore empty liposomes are provided in an amount effective to reduce thecardiac channelopathies or conditions resulting from irregularities oralterations in cardiac patterns, and the empty liposomes consist of atleast one liposome selected from at least one of phosphatidylserine,phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid,cerebrosides, dicetylphosphate, dipalmitoyl-phosphatidylglycerol,stearylamine, dodecylamine, hexadecyl-amine, acetyl palmitate, glycerolricinoleate, hexadecyl sterate, isopropyl myristate, amphoteric acrylicpolymers, fatty acid, fatty acid amides, diacylglycerol,diacylglycerolsuccinate, DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine), or DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]).
 2. Thecomposition of claim 1, wherein the composition is used for thetreatment or prevention of non-cardiac diseases of hERG.
 3. Thecomposition of claim 1, wherein the composition is adapted for enteral,parenteral, intravenous, intraperitoneal, or oral administration.
 4. Thecomposition of claim 1, wherein the active agent and the one or moreempty liposomes may be bound or conjugated together.
 5. The compositionof claim 1, wherein the one or more empty liposomes consist essentiallyof anionic, cationic, or neutral lipids.
 6. The composition of claim 1,wherein the composition further comprises a pharmaceutically acceptabledispersion medium, solvent, or vehicle, wherein the active agent, theone or more empty liposomes or both are dissolved, dispersed, orsuspended in the medium, the solvent, or the vehicle.
 7. A compositionfor preventing or treating one or more adverse reactions arising fromadministration of a therapeutically active agent or a drug in a humanthat inhibits the activity of an ether-a-go-go-related gene (hERG)consisting of: one or more pharmacologically active agents provided inan amount that causes at least one of IKr channel inhibition or QTprolongation and one or more empty liposomes, wherein the one or morepharmacologically active agents are kinase inhibitors selected fromcrizotinib and nilotinib, wherein the amount of the one or morepharmacologically active agents is sufficient to treat a cancer, whereinthe cancer is selected from non-small cell lung cancer or chronicmyeloid leukemia; and one or more empty liposomes are administered priorto, concomitantly, or after administration of the therapeutically activeagent or the drug in an amount effective to reduce the adverse reactionsarising from administration of the therapeutically active agent or drug,and the one or more empty liposomes consist essentially of one or morelipids selected from phosphatidylserine, phosphatidylinositol,sphingomyelin, cardiolipin, phosphatidic acid, cerebrosides,dicetylphosphate, dipalmitoyl-phosphatidylglycerol, stearylamine,dodecylamine, hexadecyl-amine, acetyl palmitate, glycerol ricinoleate,hexadecyl sterate, isopropyl myristate, amphoteric acrylic polymers,fatty acid, fatty acid amides, diacylglycerol, diacylglycerolsuccinate,DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), or DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]).
 8. Thecomposition of claim 7, wherein the therapeutically active agent or adrug is used in a prevention or a treatment of one or more cardiac ornon-cardiac diseases in the human or animal subject.
 9. The compositionof claim 7, wherein the composition is used for the treatment orprevention of non-cardiac related diseases of hERG.
 10. The compositionof claim 7, wherein the composition is adapted for enteral, parenteral,intravenous, intraperitoneal, or oral administration.
 11. Thecomposition of claim 7, wherein the active agent and the one or moreempty liposomes may be bound or conjugated together.
 12. The compositionof claim 7, wherein the one or more empty liposomes consist essentiallyof anionic, cationic, or neutral lipids.
 13. The composition of claim 7,wherein the one or more empty liposomes comprise DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-rac-[1-glycerol]).